Receptor gene screening for detecting or diagnosing cancer

ABSTRACT

Compositions and methods that use the body&#39;s natural secretory immune system in a new way against steroid hormone responsive tumors of the breast and prostate, as well as other glandular/mucus epithelial tissues such as colon, ovary, endometrium, kidney, bladder, stomach, pancreas and secretory pituitary gland are provided. Also provided are new ways of identifying carcinogenic, or potentially carcinogenic, bacteria in a tissue or body fluid to provide better anti-cancer therapies and preventatives than have been available previously.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/852,547, filed on May 10, 2001, now U.S. Pat. No. 7,947,463, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.60/231,273, filed Sep. 8, 2000; U.S. Provisional Patent Application Ser.No. 60/229,071, filed Aug. 30, 2000; U.S. Provisional Patent ApplicationSer. No. 60/208,111, filed May 31, 2000; and U.S. Provisional PatentApplication Ser. No. 60/208,348, filed May 31, 2000, all of which arehereby incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Research leading to the present invention was supported in part by thefederal government under Grant Nos. DAMD17-94-J-4473, DAMD17-98-8337 andDAMD17-99-1-9405 awarded by the Defense Department through the US ArmyMedical Research and Materiel Command, Breast Cancer Research Program.The United States government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to risk assessment, detection,diagnosis, prognosis, treatment and prevention of steroid hormoneresponsive cancers of mucosal epithelial tissues (i.e., glands andtissues that secrete or are bathed by secretory immunoglobulins). Moreparticularly, the invention relates to negative (inhibitory) regulationof steroid hormone responsive cancer cell proliferation, and to theimmunoglobulin inhibitors and the receptors that mediate suchregulation.

2. Description of Related Art

Finding a naturally occurring biochemical defense mechanism capable ofcontrolling neoplastic growth has been the goal of a number ofresearchers for many years. Use of the immune system against malignanttumors forms the basis for many anti-cancer strategies. For example,U.S. Pat. No. 5,980,896 describes certain antibodies, antibody fragmentsand antibody conjugates and single-chain immunotoxins directed againsthuman carcinoma cells. Conventional anti-tumor immunotherapies rely onantibody-antigen recognition chemistry, and on targeting of antibodiesagainst various antigenic features of tumor cells in order to triggerdestruction of the tumor cells by the body's immune system or to targetthe tumor cells with antibody conjugates of various cytotoxic orchemotherapeutic agents. In practice, however, tumors in vivo havegenerally not been found to be very immunogenic and in many instancesappear to be capable of evading the body's immune response. Today agreat deal of anti-cancer work is directed at finding ways of increasingthe immunogenicity of a tumor cell in vivo. For example, U.S. Pat. No.6,120,763 (Fakhrai et al.) describes a method of preventing or reducingthe severity of a cancer in a subject by stimulating the subject'simmune response against the cancer. Many studies have attempted use ofIgG as passive immunity or stimulation of natural IgG production torestrict tumor growth. As of today, there are no known vaccines forbreast cancer, prostate cancer, or any other forms of mucosal cancers(Smyth M J et al. (2001) Nature Immunol 2, 293-299).

There is a second type of immune system that is very important to thefunction and protection of the body. The immunological function andphysiological properties of the body's secretory immune system has beenrecognized for many years (Tomasi T B et al. (1965) J Exp Med 121,101-124; Brandtzaeg P and Baklien K (1977) Ciba Foundation Symposium 46,77-113; Tomasi T B (1970) Ann Rev Med 21, 281-298; Spiegelberg H L(1974) Adv Immunol 19, 259-294; Tomasi T B (1976) The Immune System ofSecretions, Prentice-Hall, Englewood Cliffs, N.J.; Mestecky J and McGheeJ R (1987) Adv Immunol 40, 153-245). It was established thatimmunoglobulin A (IgA) represents 5 to 15% of the total plasmaimmunoglobulins in humans (Spiegelberg H L (1974) Adv Immunol 19,259-294). IgA has a typical immunoglobulin four-chain structure (M_(r)160,000) made up of two heavy chains (M_(r) 55,000) and two light chains(M_(r) 23,000) (Fallgreen-Gebauer E et al (1993) Biol Chem Hoppe-Seyler374, 1023-1028; Kratzin H et al. (1978) Hoppe-Seylers Z Physiol Chem359, 1717-1745; Yang C et al. (1979) Hoppe-Seylers Z Physiol Chem 360,1919-1940; Eiffert H et al. (1984) Hoppe-Seylers Z Physiol Chem 365,1489-1495). In humans, there are two subclasses of IgA. These are IgA1and IgA2 that have 1 and 2 heavy chains, respectively. The IgA2 subclasshas been further subdivided into A₂m(1) and A₂m(2) allotypes (Mestecky Jand Russell M W (1986) Monogr Allergy 19, 277-301; Morel A et al. (1973)Clin Exp Immunol 13, 521-528). IgA can occur as monomers, dimers,trimers or multimers (Lüllau E et al. (1996) J Biol Chem 271,16300-16309). In plasma, 10% of the total IgA is polymeric while theremaining 90% is monomeric. Formation of dimeric or multimeric IgArequires the participation of an elongated glycoprotein of approximatelyM_(r) 15,000 designated the “3” chain (Mestecky J et al. (1990) Am J Med88, 411-416; Mestecky J and McGhee J R (1987) Adv Immunol 40, 153-245;Cann G M et al. (1982) Proc Natl Acad Sci USA 79, 6656-6660).Structurally, the J chain is disulfide linked to the penultimatecysteine residue of heavy chains of two IgA monomers to form a dimericcomplex of approximately M_(r) 420,000. The general structure of thedimer has been well described in the literature (Fallgreen-Gebauer E etal (1993) Biol Chem Hoppe-Seyler 374, 1023-1028). Multimeric forms ofIgA and IgM require only a single J chain to form (Mestecky J and McGheeJ R (1987) Adv Immunol 40, 153-245; Chapus R M and Koshland M E (1974)Proc Natl Acad Sci USA 71, 657-661; Brewer J W et al. (1994) J Biol Chem269, 17338-17348). The structures and chemical properties of IgA and IgMhave been described in detail (Janeway C A Jr et al. (1996)Immunobiology, The Immune System in Health and Disease, Second edition,Garland Publishing, New York, pp 3-32 and pp 8-19).

Dimeric and multimeric IgA and IgM are secreted by a number of exocrinetissues. IgA is the predominant secretory immunoglobulin present incolostrum, saliva, tears, bronchial secretions, nasal mucosa, prostaticfluid, vaginal secretions, and mucous secretions from the smallintestine (Mestecky J et al. (1987) Adv Immunol 40, 153-245; Goldblum RM, et al. (1996) In: Stiehm E R, ed, Immunological Disorders in Infantsand Children, 4^(th) edition, Saunders, Philadelphia, pp 159-199;Heremans J F (1970) In: Immunoglobulins, Biological Aspects and ClinicalUses, Merler E, ed, National Academy of Sciences, Wash DC pp 52-73;Tomasi T B Jr (1971) In: Immunology, Current Knowledge of Basic Conceptsin Immunology and their Clinical Applications, Good R A and Fisher D W,eds, Sinauer Associates, Stanford, Conn., p 76; Brandtzaeg P (1971) ActaPath Microbiol Scand 79, 189-203). IgA output exceeds that of all otherimmunoglobulins, making it the major antibody produced by the body daily(Heremans J F (1974) In: The Antigens, Vol 2, Sela M, ed, AcademicPress, New York, pp 365-522; Conley M E et al. (1987) Ann Intern Med106, 892-899. IgA is the major immunoglobulin found in humanmilk/whey/colostrum (Ammann A J et al. (1966) Soc Exp Biol Med 122,1098-1113; Peitersen B et al. (1975) Acta Paediatr Scand 64, 709-717);Woodhouse L et al. (1988) Nutr Res 8, 853-864). IgM secretion is lessabundant but can increase to compensate for deficiencies in IgAsecretion. J chain containing IgA is produced and secreted by plasma Bimmunocytes located in the lamina propria just beneath the basementmembrane of exocrine cells (Brandtzaeg P (1985) Scan J Immunol 22,111-146). The secreted IgA binds to a M_(r) 100,000 poly-Ig receptorpositioned in the basolateral surface of most mucosal cells (Heremans JF (1970) In: Immunoglobulins, Biological Aspects and Clinical Uses,Merler E, ed, National Academy of Sciences, Wash DC, pp 52-73;Brandtzaeg P (1985) Clin Exp Immunol 44, 221-232; Goodman J W (1987) In:Basic and Clinical Immunology, Stites D P, Stobo J D and Wells J V, eds,Appleton and Lange, Norwalk, Conn., Chapter 4). The receptor-IgA complexis next translocated to the apical surface where IgA is secreted. Thebinding of dimeric IgA to the poly-Ig receptor is completely dependentupon the presence of a J chain (Brandtzaeg P (1985) Scan J Immunol 22,111-146; Brandtzaeg P and Prydz H (1984) Nature 311:71-73; Vaerman J-Pet al. (1998) Eur J Immunol 28, 171-182). Monomeric IgA will not bind tothe receptor. The J chain requirement for IgM binding to the poly-Igreceptor is also true for this immunoglobulin (Brandtzaeg P (1985) ScanJ Immunol 22, 111-146; Brandtzaeg P (1975) Immunology 29, 559-570;Norderhaug I N et al. (1999) Crit. Rev Immunol 19, 481-508). Because IgAand IgM bind to the poly-Ig receptor via their Fc domains, and becauseof a repeating Ig-like structure in the extracellular domains, thepoly-Ig receptor classifies as a member of the Fc superfamily ofimmungobulin receptors (Kraj{hacek over (c)}i P et al. (1992) Eur JImmunol 22, 2309-2315; Daëron M (1997) Annu Rev Immunol 15, 203-234).

During passage of IgA through the cell, its structure is modified. AM_(r) 80,000 fragment of the receptor containing all five of theextracellular domains becomes covalently attached to dimeric IgA to formsecretory IgA (sIgA) (Fallgreen-Gebauer E et al (1993) Biol ChemHoppe-Seyler 374, 1023-1028). The receptor that mediates thetranslocation has been interchangeably called the “poly-Ig receptor”(poly-Ig receptor) or the “secretory component” (Kraj{hacek over (c)}i Pet al. (1992) Eur J Immunol 22, 2309-2315). Except where notedotherwise, for the purposes of the present disclosure, the term “poly-Igreceptor” refers to the full length M_(r) 100,000 transmembrane proteinand the term “secretory component” denotes only the M_(r) 80,000extracellular five domains of the receptor that become covalentlyattached to IgA in forming the sIgA structure (Fallgreen-Gebauer E et al(1993) Biol Chem Hoppe-Seyler 374, 1023-1028; Kraj{hacek over (c)}i P etal. (1992) Eur J Immunol 22, 2309-2315). Because of the unique structureof sIgA, it is highly resistant to acid and proteolysis (Lindh E (1975)J Immunol 114, 284-286) and therefore remains intact in secretions toperform extracellular immunological functions. IgM also binds secretorycomponent, but not covalently (Lindh E and Bjork I (1976) Eur J Biochem62, 271-278). However, IgM is less stabilized because of its differentassociation with the secretory component, and therefore has a shorterfunctional survival time in acidic secretions (Haneberg B (1974) Scand JImmunol 3, 71-76; Haneberg B (1974) Scand J Immunol 3, 191-197). IgA andIgM are known to bind to bacterial, parasite and viral surface antigens.These complexes bind to receptors on inflammatory cells leading todestruction of the pathogen by antibody-dependent cell-mediatedcytotoxicity (Hamilton R G (1997) “Human immunoglobulins” In: Handbookof Human Immunology, Leffell M S et al., eds, CRC Press, Boca Raton,Chapter 3).

The major immunoglobulins secreted as mucosal immune protectors includeIgA, IgM and IgG. In human serum, the percent content of IgG, IgA andIgM are 80, 6 and 13%, respectively. In humans, the major subclasses ofIgG are IgG1, IgG2, IgG3 and IgG4. These are 66, 23, 7 and 4% of thetotal. IgG, respectively. The relative content of human immunoglobulinclasses/subclasses in adult serum follow the orderIgG1>IgG2>IgA1>IgM>IgG3>IgA2>IgD>IgE (Spiegelberg H L (1974) Adv Immunol19, 259-294). When the serum concentrations of immunoglobulins arecompared to those in exocrine secretion fluids, the relative contentschange dramatically (Brandtzaeg P (1983) Ann NY Acad Sci 409, 353-382;Brandtzaeg P (1985) Scand J Immunol 22, 111-146). For example incolostrum (a breast fluid secretion), IgA is ≧80% of the totalimmunoglobulins. IgM is ≦10% of the total. IgG represents a few percent.In human colostrum and milk, IgG1 and IgG2 are the major subclasses ofIgG (Kim K et al. (1992) Acta Paediatr 81, 113-118). Clearly, comparisonof serum and mucosal fluid concentrations indicate selectiveimmunoglobulin secretion. The secretion mechanism for IgA and IgM arewell described. Conversely, there is a fundamental question surroundingIgG secretion. There is no “J” chain present in IgG1 and IgG2. From theknown facts of transcytosis/secretion of immunoglobulins (Johansen F Eet al. (2000) Scand J Immunol 52, 240-248), it is unlikely that IgGsecretion is mediated by the poly-Ig receptor. An epithelial receptorspecific for IgG1 has been reported in bovine mammary gland (Kemler R etal. (1975) Eur J Immunol 5, 603-608). Apparently, it preferentiallytransports this class of immunoglobulins from serum into colostrum.Despite this 1975 report however, the receptor has not been chemicallyor structurally identified nor has the mechanism of transport of IgGmonomers been satisfactorily defined. Certainly no growth function wasascribed to this “IgG1 receptor” in the 1975 Kemler et al. report. It ispossible that this receptor is a member of a large group now designatedas Fc receptors (Fridman W H (1991) FASEB J 5, 2684-2690), but there isone study with IgG showing that of 31 different long-term humancarcinoma cell lines including breast “all lines were found to beconsistently Fc receptor negative” (Kerbel R S et al. (1997) Int JCancer 20, 673-679). One possible candidate for the epithelial transportof IgG1 is the neonatal Fc receptor (Raghavan M and Bjorkman P J (1996)Annu Rev Cell Dev Biol 12, 181-220). However, there is no indication yetof the presence of this receptor in adult mucosal tissues.

All human mucus membranes are protected by the secretory immune system(Hanson L Å and Brandtzaeg P (1989) In: Immunological Disorders inInfants and Children, 3^(rd) edition, Stiehm E R, ed, Saunders,Philadelphia, pp 169-172). The primary protector is sIgA that isproduced as dimers and larger polymers. A single joining “J” chainconnects IgA monomers to form the dimers and polymers (Garcia-Pardo A etal. (1981) J Biol Chem 256, 11734-11738), and connects monomers of IgMto give pentamers (Niles M J et al. (1995) Proc Natl Acad Sci USA 92,2884-2888). This critical joining endows these structures with a veryimportant immunological property. Dimeric and polymeric sIgA have a highantigen binding valence that effectively agglutinates/neutralizesbacteria and virus (Janeway C A Jr et al. (1999) Immunobiology, TheImmune System in Health and Disease, 4^(th) edition, Garland Publishing,New York, pp. 326-327). Also, sIgA shows little or no complementactivation. This means that it does not cause inflammatory responses(Johansen F E et al. (2000) Scand J Immunol 52, 240-248). In addition,the fact that IgA exists as two separate forms is significant (Loomes LM et al (1991) J Immunol Methods 141, 209-218). The IgA1 predominates inthe general circulation. In contrast, IgA2 is often higher in mucosalsecretions such as those from breast, gut, and respiratory epithelium,salivary and tear glands, the male and female reproductive tracts, andthe urinary tracts of both males and females. This difference inproportions is important to immune protection of mucosal surfaces.Although the secretory form of IgA1 is by and large resistant toproteolysis (Lindh E (1975) J Immunol 114, 284-286), a number ofdifferent bacteria secrete proteolytic enzymes that cleave it into Faband Fc fragments (Warm J H et al. (1996) Infect Immun 64, 3967-3974;Poulsen K et al. (1989) Infect Immun 57, 3097-3105; Gilbert J V et al.(1988) Infect Immun 56, 1961-1966; Reinholdt J et al. (1993) InfectImmun 61, 3998-4000; Blake M S and Eastby C (1991) J Immunol Methods144, 215-221; Burton J et al. (1988) J Med Chem 31, 1647-1651; MortensenS B and Kilian M (1984) Infect Immun 45, 550-557; Simpson D A et al.(1988) J Bacteriol 170, 1866-1873; Blake M S and Swanson J et al. (1978)Infect Immun 22, 350-358; Labib R S et al. (1978) Biochim Biophys Acta526, 547-559). In effect, the bacterial proteinases negate theneutralizing effects of multivalent sIgA1. In contrast, because ofstructural differences (Chintalacharuvu K R and Morrison S L (1996) JImmunol 157, 3443-3449), IgA2 lacks sites required for proteolysis. Thismakes IgA2 more resistant to bacterial digest than IgA1 (Hamilton R G(1997) “Human immunoglobulins” In: Handbook of Human Immunology, LeffellM S et al., eds, CRC Press, Boca Raton, Chapter 3). With regard to IgM,its function is somewhat different. IgM antibodies serve primarily asefficient agglutinating and cytolytic agents. They appear early in theresponse to infection and are largely confined to the bloodstream.Whether secreted or plasma-borne, IgM is a highly effective activator ofthe classical complement cascade. It is less effective as a neutralizingagent or an effector of opsinization (i.e. facilitation of phagocytosisof microorganisms). Nonetheless, IgM complement activation causes lysisof some bacteria. The effects of the IgG class are more encompassing.All four subclasses cause neutralization, opsinization and complementactivation to defend against mucosal microorganisms. IgG1 is an activesubclass in this regard (Janeway C A Jr et al. (1999) Immunobiology, TheImmune System in Health and Disease, 4^(th) edition, Garland Publishing,New York, pp 326-327).

With regard to breast cancer and prostate cancer etiology, there hasbeen only limited attention given to the role of the immune system.Other issues have been considered more important for placing individualsin the at-risk groups for developing cancer in general and breast cancerspecifically. This has led to searches for risk factors. Advancescertainly have been made. We now have the benefit of the investment ofscientific effort and volume of new information that was obtained.Breast cancer is one useful example of our advances. There have beenseveral reviews of this topic published since 1979 (Kelsey J L (1979)Epidemiol Rev 1, 74-109; Kelsey J L and Berkowitz G S (1988) Cancer Res48, 5615-5623; Kelsey J L and Gammon M D (1990) Epidemiol Rev 12,228-240; Colditz G A (1993) Cancer 71, 1480-1489; Alberg A J andHelzlsouer K J (1997) Current Opinion Oncology 9, 505-5111). Althoughreproductive factors, body build, oral contraceptives, estrogenreplacement therapy, diethylstilbestrol, hormonal imbalances, diet(particularly high fat consumption), alcohol consumption, radiation,familial aggregation and heredity have been studied, and some of theseidentified as risk factors, there remains no known cause of the 70% ormore of breast cancers now known as “sporadic” because they appear tooccur randomly in the population and certainly without any known geneticpattern. Plainly stated, for the vast majority of women who developbreast cancer, there is no known genetic cause. Even with the bestapplications of the epidemiology cited above, the answer has not beenforthcoming for this majority.

The only cases where there is a defined genetic origin of breast cancerinvolve the BRCA1 and BRCA2 genes. The BRCA1 gene has been cloned,sequenced and localized to chromosome 17 (Hall J M et al. (1990) Science(Wash DC) 250, 1684-1689; Bowcock A M (1993) Breast Cancer Res Treat 28,121-135; Mild Y et al. (1994) Science (Wash DC) 266, 66-71). Anothergene, BRCA2, has also been identified and linked to chromosome 13q(Wooster R et al. (1995) Nature (Lond) 378, 789-792; Tavigian S V et al.(1996) Nature Genet. 12, 333-337). BRCA1 gene lesions are linked tobreast and ovarian cancer. BRCA2 is more associated with ovarian cancerthan breast cancer. Together, these two genes are thought to account formost of the inheritable/familial breast cancer in the United States(Krainer M et al. (1997) New Eng J Med 336, 1416-1421). However, oneimportant fact that must be recognized is that these genes are probablycarried by fewer than 400 women in the United States and therefore areresponsible for a relatively small number of human breast cancers (KingM-C et al (1993) JAMA 269, 1975-1980; Biesecker B B et al. (1993) JAMA269, 1970-1974). Although these genes continue to be studiedintensively, it is far from clear that they have a significant causativerole in the 70% or more of “sporadic” non-inherited breast cancers. Infact, the essential point is that the origin of the vast majority ofbreast cancers remains unknown.

Currently these two genes, BRCA1 (Lynch H et al. (1978) Cancer 41,1543-1549; Hall J M et al. (1990) Science (Wash DC) 2500684-1689; NarodS A et al. (1991) Lancet 338, 82-83; Steichen-Gersdorf E et al. (1994)Am J Hum Genet. 55, 870-875; Mild Y et al. (1994) Science (Wash DC) 266,66-71; Smith S et al. (1992) Nature Genet. 2, 128-131) and BRCA2(Wooster R et al. (1994) Science (Wash DC) 265, 2088-2090; Wooster R etal. (1995) Nature 378, 789-792), have been related to early onset offamilial (autosomal dominant) breast and ovarian cancer. In contrast toBRCA1, which is linked predominantly to female cancers, BRCA2 is alsolinked to male breast cancer. As pointed out above, about 1% of thebreast cancers occurring in the United States are related to those genes(Easton F D et al. (1994) Lancet 344, 761). Their gene sequences havebeen fully characterized and in the case of BRCA1, many mutations havebeen identified (Shattuck-Eidens D et al. (1995) JAMA 273, 535-552;Simard J et al. (1994) Nature Genet. 8, 392-398; Castilla L H et al.(1994) Nature Genet. 8, 387-391). Mutations in these genes wereinitially considered to confer more than 80% lifetime risk fordeveloping breast and/or ovarian cancer (Easton D F et al. (1993) Am JHum Genet. 52, 678-701). More recent results have reduced the roles ofBRCA1 and BRCA2 in breast cancers (Struewing J P et al. (1997) New Eng JMed 336, 1401-1408; Couch F J et al. (1997) New Eng J Med 336,1409-1415; Krainer M et al. (1997) New Eng J Med 336, 1416-1421). BRCA1and BRCA2 may have roles in sporadic breast and ovarian cancers, but towhat extent is open to question (Futreal P A et al. (1994) Science (WashDC) 266, 120-122; Merajver S D et al. (1995) Nature Genet. 9, 439-443).In addition to BRCA1 and BRCA2, the tumor suppressor gene p53 has beenimplicated in both familial (germ line) and sporadic breast cancers(Malkin D et al. (1990) Science (Wash DC) 250, 1233-1238; Coles C et al.(1992) Cancer Res 52, 5291-5298; Elledge R M and Allred D C (1994)Breast Cancer Res Treat 32, 39-47). However, this genetic link accountsfor at most 25% of breast cancers. It is possible that germ linemutations in p53 also are related to a fraction of prostate cancers(Malkin D et al. (1990) Science (Wash DC) 250, 1233-1238). One area ofactive investigation focuses on the 70% of breast cancers termed“sporadic,” because they are not familial and not related to anycurrently known epidemiological risk factor. An effective means ofassessing genetic risk for sporadic breast cancers, prostate cancers,and other cancers of glandular/mucosal epithelial tissues, simply doesnot exist today in the conventional medical arsenal against cancer.

The genetic origin of prostate cancers has been even more elusive thanthat of breast cancers. Although a gene for prostate cancersusceptibility has been localized to chromosome 17q, it does not appearto be related to BRCA1 (PCT Pub. App. No. WO0027864). Other prostatecancer susceptibility genes have been localized to chromosomes 13q(Cooney K A et al. (1996) Cancer Res 56, 1142-1145) and to chromosomes8p, 10q and 16q (Veronese M L et al. (1996) Cancer Res 56, 728-732).From the data available, it is clear that the genetic origin of prostatecancer has not been identified. This fact alone opens the issue ofcause. While genetic analysis will continue to be important, it will notprovide the essential information about what is causing breast andprostate cancer.

In a conceptually different approach to identifying cancer-relatedgenes, Dr. Ruth Sager has suggested a departure from the conventionalavenues of identifying cancer-related genes by searching for mutations(Class I genes), and instead or additionally focusing on the role ofexpression genetics in cancer (Class II genes) (Sager R (1997) Proc NatlAcad Sci 94, 952-955). Dr. Sager has proposed that far more genes aredown regulated at the transcriptional level in cancer cells than aremutated and that crucial “oncogenic” molecules may not be mutated.Consistent with that proposition others have reported (Thompson M F etal. (1995) Nature Genet. 9, 444-450) that reduced amounts of BRCA1 mRNA,representing down-regulation of the wild-type gene, were found inprimary tumors of the nonfamilial disease. Characterization of othergenes whose expression is altered in cancer cells, and understandingtheir functions, will provide penetrating insight into the regulatoryinteractions that have been upset in cancer.

With regard to the origins of mucosal cancer, and especially breast andprostate, there has been little advance. In general, it is thought thatenvironmental carcinogens are the origin. However, this has yet to beproven. Another familiar concept is the idea that bacteria may beinvolved in carcinogenesis (oncogenesis). For example, see Parsonnet J(1995) Environ Health Perspect 103 (Suppl), 263-268; Mackowiak P A(1987) Am J Med 82, 79-97; Cassell G H (1998) Emerg Infect Dis 4,475-487; Nauts H C (1989) Cancer Surv 8, 713-723; Venitt S (1996)Environ Health Perspect 104 (Suppl), 633-637; Miller J H (1996) CancerSurv 28, 141-153; Buiuc D and Dorneanu O (1989) Rev Med Chir Soc Med NatIasi 93, 223-227).

Involvement of bacteria, or other infectious agents, in some types oflymphoid cancers such as Hodgkin's disease and leukemia has beensuggested (Comment of Editor: Infective cause of childhood leukaemia(1989) Lancet 1 (1829), 94-95; Serraino D et al. (1991) Int J Cancer 47,352-357; Glaser S L and Jarrett R F Baillieres (1996) Clin Haematol 9,401-416; Wolf J and Diehl V (1994) Ann Oncol 5 (Suppl 1), 105-111).

Studies suggesting that Helicobacter pylori is directly causative ingastric cancer have recently been described. H. pylori is the onlybacterium known to date to have been classified as a Class I carcinogenby the International Agency for Research on Cancer (IARC). Thisclassification indicates that by generally accepted scientific standards(Nyren O (1998) Semin Cancer Biol 8, 275-283) this microorganism is nowgenerally considered to be a causative factor in development of gastriccancers in infected humans. Recently it has been reported that Chlamydiatrachomatis infection is strongly associated with subsequent developmentof invasive cervical squamous cell carcinoma (Anttila T et al. (2001)JAMA 283, 47-51). The possibility that bacteria are involved in largebowel/colon cancer has also been mentioned (McBurney M I et al. (1987)Nutr Cancer 10, 23-28), however no firm conclusions have been reached asyet.

Finally, the issue of prevention deserves special comment. There are noknown methods of preventing cancer other than observing life stylechanges and environmental changes that place individuals in the low riskgroups. Tamoxifen has been considered as a potential “prevention” forbreast cancer in high risk women, but as yet has not been widelyaccepted because of the physiologic and endocrine aberrations caused bythis agent when used long term. In short, even though prevention isremarkably pressing, there has been a dearth of studies of new methodsthat do not disrupt the normal lifestyles and reproductive capacity ofwomen.

Conventional immunological approaches to treating malignant tumors havegenerally proven inadequate. In addition, except for recent advanceswith respect to Helicobacter pylori and Chlamydia trachomatis (Anttila Tet al. (2001) JAMA 283:47-51), anti-bacterial approaches for combatingthe cause(s) of malignant transformation do not appear promising.Relying only on the existing technologies, effective diagnostic andtherapeutic agents, treatments and preventatives for widespread use inbreast and prostate cancers, and cancers of other glandular/mucosalepithelial tissues, do not appear to be on the near horizon.

SUMMARY OF THE INVENTION

New methods and compositions for use against steroid hormone responsivetumors of the breast and prostate, as well as against tumors of otherglandular/mucus epithelial tissues such as colon, ovary, endometrium,kidney, bladder, stomach, pancreas and secretory pituitary gland areprovided which are based on previously unrecognized activities ofcertain components of the body's natural immune system. References inthis disclosure to the “new natural immune mechanism” or the “newimmunotherapies,” refer to the previously unrecognized cell growthinhibitory function of certain constituent parts of the secretory immunesystem, particularly dimeric/polymeric IgA, polymeric IgM and IgG1, asdistinguished from the well known functions of those immunoglobulinsbased on antigen-antibody recognition. For the purposes of thisdisclosure, the term “cell growth” refers to cell proliferation or anincrease in the size of a population of cells rather than merely to anincrease in cytoplasmic volume of an individual cell. The term “steroidhormone responsive” cell refers to a cell that requires the binding of asteroid hormone to a steroid hormone binding receptor in the cell inorder for that cell to be stimulated to grow (i.e., proliferate). Forexample, normal ductile cells in the pubescent breast are estrogenresponsive or stimulated by estrogen to proliferate. ER⁺ breast cancercells also possess a functional estrogen binding receptor and are alsoestrogen responsive. By contrast, ER⁻ breast cancer cells do not have afunctional estrogen receptor and demonstrate autonomous cell growth,i.e., they are stimulated to proliferate without the influence of asteroid hormone. New ways of identifying carcinogenic, or potentiallycarcinogenic, bacteria in a tissue or body fluid are also provided, and,individually or together with the above-described newimmunotechnologies, are expected to provide or aid in widespreadimplementation of better anti-cancer therapies and preventatives thanhave been available previously.

In accordance with certain embodiments of the present invention, methodsof assessing risk or susceptibility of an individual to developing aneoplastic lesion or cancerous tumor of a mucosal epithelial tissue areprovided. In some embodiments the method includes detecting, and in somecases also quantitating, a steroid hormone reversible immunoglobulininhibitor of steroid hormone responsive cell growth in a body fluid orsecretion obtained from said subject, such as serum, plasma, colostrum,breast aspirates, saliva, tears, bronchial secretions, nasal mucosa,prostatic fluid, urine, semen or seminal fluid, vaginal secretions,ovarian aspirates, stool, and mucous secretions from the small intestineor stomach. The absence or deficiency of the immunoglobulin inhibitorcompared to a predetermined standard material indicates or suggests thata steroid hormone responsive mucosal epithelial tissue in the body ofthe individual is secreting or bathed by less than a cell growthinhibitory amount of the immunoglobulin inhibitor. For the purposes ofthis disclosure, the term “immunoglobulin inhibitor” refers to asecretory immunoglobulin, preferably one or more of the secretoryimmunoglobulins IgA, IgM and IgG1, that is active for inhibitingproliferation of a steroid hormone responsive cancer cell maintained ina suitable nutrient medium under cell growth promoting conditions, inthe absence of an inhibition-reversing amount of the steroid hormone orother substance that mimics this steroid hormone effect. Theimmunoglobulin inhibitory activity, also referred to as immunoglobulininhibition, is distinct from any additional antigen-antibody recognitionbased immunological functions of the immunoglobulin inhibitors. The term“steroid hormone reversible immunoglobulin inhibitor” refers to thecharacteristic of the preferred immunoglobulin inhibitors that theircell growth inhibitory activity is steroid hormone reversible. “Cellgrowth promoting conditions” refer to favorable environmentalconditions, other than defined medium components, and include suchthings as gaseous environment, humidity, temperature, pH, and the like.For example, cell growth promoting conditions could include incubationat 37° C. in a humid atmosphere of 5% (v/v) CO₂ and 95% (v/v) air in adefined nutrient medium at pH 7.4.

In certain embodiments the risk assessment method includes measuring theamount and/or activity of an immunoglobulin inhibitor in a specimencomprising a defined amount of body fluid or secretion from theindividual. In certain preferred embodiments the method includessubstantially depleting steroid hormone from the fluid specimen to yielda steroid hormone depleted specimen, and then assaying an aliquot ofthat hormone depleted specimen for detecting or measuring steroidhormone reversible inhibition of steroid hormone responsive cancer cellproliferation.

Some embodiments of the risk assessment method include the followingassay protocol: (a) maintaining a predetermined population of steroidhormone-responsive cells in a ferric ion-free, calcium ion-containing,serum-free nutrient medium, the cells being serum free and obtained froma steroid hormone-responsive cell line; (b) adding a predeterminedamount of the steroid hormone to the medium, the amount being sufficientto stimulate cell growth under cell growth promoting conditions; (c)adding a predetermined amount of a steroid hormone depleted specimen ofa body fluid or secretion to the medium, to yield a test mixture; (d)incubating the test mixture for a predetermined period of time undercell growth promoting conditions; (e) measuring the cell population inthe test mixture after the predetermined period of time; (f) measuringthe cell population in a control incubation mixture like the testmixture, except lacking an amount of the specimen. Preferably anycytotoxic effects of the specimen are also measured. The differencebetween the cell populations before and after the incubation period isdetermined, a significant increase in the population indicating theabsence of inhibition of cell growth by that amount of specimen in thepresence of the selected amount of steroid hormone. A significant lackof increase in the cell population, which is not attributable tocytotoxic effects of the specimen, is an indicator of inhibition of cellgrowth by the inhibitors contained in the specimen when tested in thepresence of a given concentration of steroid hormone. In preferredembodiments the assay also includes detecting or determining steroidhormone reversibility of the specimen's inhibitory activity in thepresence of a predetermined increased amount of steroid hormone.

Also provided in accordance with certain embodiments of the presentinvention are an in vitro method of, detecting loss of immunoglobulinregulation of steroid hormone responsive cell growth. In certainembodiments the method comprises assaying for inability of a mucosalepithelial cell to bind at least one of the immunoglobulins IgA, IgM andIgG1.

Certain other embodiments of the invention provide a method of detectinga mediator of immunoglobulin inhibition of steroid hormone responsivecell growth that includes detecting a poly-Ig receptor in a mucosalepithelial cell.

Certain other embodiments of the invention provide a method of detectinga gene coding for a mediator of immunoglobulin inhibition of steroidhormone responsive cell growth that includes detecting the presence of apoly-Ig receptor gene in a mucosal epithelial cell.

Still other embodiments of the invention provide a method of detecting agenetic defect in a gene coding for a mediator of immunoglobulininhibition of steroid hormone responsive cell growth comprisingscreening a genomic or cDNA library of a mucosal epithelial cell for adefect in a poly-Ig receptor gene.

Some embodiments of the present invention provide a method of detectingexpression of a defective mediator of immunoglobulin inhibition ofsteroid hormone responsive cell growth in a specimen of mucosalepithelial tissue, the method including detecting a defective poly-Igreceptor or Fcγ receptor protein in said specimen. The term “defective”means that the detected protein is physically similar to the nativereceptor protein, but is incapable or less capable of mediating theimmunoglobulin cell growth inhibitory effects, compared to the nativereceptor protein. Preferably the ability to mediate cell growthinhibitory effects is measured in a cell growth assay as describedelsewhere herein.

In accordance with certain other embodiments of the present invention,methods to aid in predicting increased susceptibility of a mammaliansubject to development or growth of a steroid hormone responsive cancerin a mucosal epithelial tissue is provided. In some embodiments, themethod comprises detecting the loss or impairment of negative regulationof breast tissue proliferation by the secretory immune system. In someembodiments, the method includes assaying a specimen of mucosalepithelial tissue obtained from the subject for the presence of apoly-Ig receptor capable of mediating immunoglobulin inhibition ofsteroid hormone responsive cell growth in a suitable in vitro cellculture assay. An absence of the receptor or absence of its activity formediating the immunoglobulin inhibition is suggestive that the tissuelacks functional mediators of immunoglobulin inhibition sufficient todeter development or growth of a steroid hormone responsive cancer ofthe mucosal epithelial tissue.

Also provided by certain embodiments of the invention is a method to aidin detecting transformation of a mucosal epithelial cell from normallysteroid hormone responsive to a steroid hormone responsive neoplastic,precancerous or cancerous condition, the method including assaying apopulation of the cells for loss or inactivity of receptors that mediateIgG1 inhibition of cell growth.

Further provided by certain embodiments of the invention are methods toaid in detecting progression of a steroid hormone responsive malignantmucosal epithelial cell to an autonomous cancer cell. In someembodiments, the method includes assaying for loss or inactivity of areceptor that mediates IgA and/or IgM inhibition of cell growth.

According to certain other embodiments of the invention, methods ofimaging a steroid hormone responsive mucosal epithelial tumor in vivo isprovided. In certain embodiments, the method includes contacting thetumor with at least one tagged or labeled monoclonal antibody raisedagainst a poly-Ig receptor, Fcγ receptor, IgA, IgM and IgG1, and thenimaging the tag.

In some embodiments of the present invention a method to aid indetecting or diagnosing cancer in a mammalian subject is provided. Themethod comprises determining in a population of neoplastic cells in amucosal epithelial tissue specimen obtained from the subject at leastone of the following conditions: (a) absence or diminution ofimmunoglobulin inhibition of steroid hormone responsive cell growth; (b)absence or diminution of at least one immunoglobulin inhibitor ofsteroid hormone responsive cell growth from a body fluid or secretionsecreted by or bathing said tissue; (c) absence or diminution of apoly-Ig receptor in said cells; (d) absence of a poly-Ig receptor genefrom said cells; (e) absence of heterozygosity for said poly-Ig receptorgene in said cells; (f) absence or diminution of a Fcγ receptor in saidcells; (g) absence of a Fcγ receptor gene from said cells; (h) absenceof heterozygosity for said Fcγ receptor gene in said cells; (i) absenceor diminution of TGFβ regulation of cell growth; (j) absence ordiminution of a TGFβ receptor in said cells; (k) absence of a TGFβreceptor gene from said cells; and (l) absence of heterozygosity forsaid TGFβ receptor gene in said cells. The absence or diminution ispreferably measured by comparison to similar determinations innon-neoplastic cells from the same patient or by comparison topredetermined standard values. The presence of at least one of thoseconditions is suggestive or indicative of the presence of a cancerous orprecancerous lesion, and an absence of one or more of the conditionssuggests or indicates absence of a cancerous or precancerous lesion inthe patient.

In accordance with certain additional embodiments of the invention, amethod to aid in staging a cancer of a mucosal epithelial tissue isprovided. The method includes testing or determining, in a specimen ofneoplastic cells obtained from the cancer, if the cells are stimulatedby a preselected steroid hormone proliferate in a suitable cell growthnutrient medium, and also determining at least one of the followingconditions: (a) in a specimen of body fluid or secretion secreted by orbathing said mucosal epithelial tissue, the lack of a cell growthinhibitory amount of at least one immunoglobulin inhibitor of steroidhormone responsive cell growth, (b) loss or diminution of a TGFβreceptor in said cells, (c) loss of a TGFβ receptor gene in said cellsin said cells, (d) loss of heterozygosity for said TGFβ receptor gene insaid cells, (e) loss or diminution of a poly-Ig receptor in said cells,(f) loss of a poly-Ig receptor gene in said cells, (g) loss ofheterozygosity for said poly-Ig receptor gene in said cells, (h) loss ordiminution of a Fcγ receptor in said cells, (i) loss of a Fcγ receptorgene in said cells, and (j) loss of heterozygosity for said Fcγ receptorgene in said cells. The loss or diminution in each case is measured bycomparison to similar determinations in non-neoplastic cells from thesame patient, or by previous values obtained from a previous test, or bycomparison to predetermined standard values. The presence of one or moreof the conditions suggests or indicates advancement of the stage of thecancer.

According to some embodiments of the present invention a method to aidin prognosis of a mammalian cancer patient is provided. This methodcomprises determining at least one of the following conditions: (a) in aspecimen of body fluid or secretion secreted by or bathing a mucosalepithelial tissue obtained from said patient, the lack of a cell growthinhibitory amount of at least one immunoglobulin inhibitor of steroidhormone responsive cell growth, (b) in a specimen of neoplastic cellsfrom said tissue, the loss or diminution of a TGFβ receptor, (c) in aspecimen of neoplastic cells from said tissue, the loss of a TGFβreceptor gene, (d) in a specimen of neoplastic cells from said tissue,the loss of heterozygosity for said TGFβ receptor gene, (e) in aspecimen of neoplastic cells from said tissue, the loss or diminution ofa poly-Ig receptor, (f) in a specimen of neoplastic cells from saidtissue, the loss of a poly-Ig receptor gene, (g) in a specimen ofneoplastic cells from said tissue, the loss of heterozygosity for saidpoly-Ig receptor gene, (h) in a specimen of neoplastic cells from saidtissue, the loss or diminution of a Fcγ receptor, (i) in a specimen ofneoplastic cells from said tissue, loss of a Fcγ receptor gene, and (j)in a specimen of neoplastic cells from said tissue, loss ofheterozygosity for said Fcγ receptor gene. The loss or diminution ineach instance is measured by comparison to similar determinations innon-neoplastic cells from the patient, and/or to the patient's previoustest results, and/or by comparison to predetermined standard values. Thepresence of one or more of the conditions is suggestive or indicative ofat least some degree of reduced prognosis of the patient, and an absenceof one or more of said conditions being suggestive or indicative of atleast some degree of favorable prognosis.

In accordance with certain other embodiments of the present invention, amethod to aid in suppressing or inhibiting malignant transformation orprogression in a steroid hormone responsive mucosal epithelial cell isprovided. The method comprises ensuring expression of a TGFβ receptor inthe cell sufficient to mediate TGFβ inhibition of neoplastic cellgrowth, and also ensuring expression of a poly-Ig receptor and/or a Fcγreceptor. In preferred embodiments, the method ensures that poly-Igreceptor is expressed in the cell sufficient to mediate IgA and/or IgMinhibition of steroid hormone responsive growth of the cell in theabsence of an inhibition reversing amount of the steroid hormone orsteroid hormone mimicking substance; and the Fcγ receptor is expressedsufficiently to mediate IgG1 inhibition of steroid hormone responsivegrowth of the cell in the absence of an inhibition reversing amount ofthe steroid hormone or steroid hormone mimicking substance. Ifexpression of one of those native receptors is lacking, it may benecessary to introduce an exogenous receptor gene using known genetransfer techniques.

In accordance with certain other embodiments of the invention, a methodof inhibiting or arresting in vivo cancer cell growth is provided. Themethod comprises contacting a steroid hormone responsive mucosalepithelial tissue with a pharmaceutical composition comprising apharmacologically acceptable carrier and at least one immunoglobulininhibitor of steroid hormone responsive cell growth chosen from thegroup consisting of IgA, IgM and IgG1, preferably dimeric or polymericIgA, polymeric IgM and IgG1. In some embodiments, the treatment methodalso includes administering an immunoglobulin inhibitor-mimickingsubstance such as tamoxifen or a metabolite thereof.

Certain other embodiments of the present invention provide a method oftreating cancer of a glandular or tissue that secretes or is bathed byan immunoglobulin, the method comprising enhancing the amount of atleast one immunoglobulin inhibitor of steroid hormone responsive cancercell growth secreted by or contacting the tissue. The inhibitor ispreferably IgA, IgM and IgG1.

According to certain embodiments of the present invention, a method oftreating cancer of a steroid hormone responsive mucosal/epithelialtissue is provided. In some embodiments, the method comprises detectingin a population of cancer calls obtained from the tissue the presence ofa poly-Ig receptor or a portion thereof. In some embodiments, the methodalso includes detecting in the population of cancer cells the presenceof ERγ. In still other embodiments, the method also includesadministering to an individual in need thereof, an effective amount ofan immunoglobulin mimicking substance (e.g., tamoxifen or a tamoxifenmetabolite) sufficient to inhibit cancer cell growth.

Although the cell growth inhibitory activity of the immunoglobulininhibitors is a function that is distinct from any additionalantibody-antigen recognition type immune activities, in some instancesconventional immunological techniques can be advantageously employed toproduce the desired inhibitors. Accordingly, in certain embodiments ofthe invention a method of inhibiting or arresting growth of a steroidhormone responsive tumor in a mammal is provided which includesadministering an immunogen to the mammal in an amount sufficient toinduce plasma and/or mucosal production of at least one secretoryimmunoglobulin inhibitor of steroid hormone responsive cell growthsufficient to inhibit steroid hormone responsive proliferation of aplurality of steroid hormone responsive cancer cells in the mammal. Insome embodiments the mode of administration is oral. In certainembodiments, the method also includes determining an age range of themammal during which the native production of the inhibitor(s) in themammal is less than a predetermined value. An age range in which thereare low concentrations of natural immunoglobulin inhibitors may presenta window of increased susceptibility to mutagenic or other carcinogenicevents. Some embodiments provide for administering the immunogen at apredetermined time such that production of the inhibitor(s) by themammal during that window of susceptibility is enhanced.

In certain other embodiments of the invention a method of inducingnatural mucosal production of cancer deterring factors is provided. Themethod comprises parenteral administration to a mammal of an amount ofsecretory immunoglobulin-stimulating antigen sufficient to induce plasmaand/or mucosal production of a predetermined steroid hormone responsivecancer cell growth inhibiting amount of at least one secretoryimmunoglobulin IgA, IgM and IgG1.

In still other embodiments of the invention a method of enhancing levelsof cancer deterring factors in a body fluid bathing a gland or mucosaltissue is provided. The method includes introducing into the body of anindividual in need thereof at least one exogenous steroid hormoneresponsive cell growth immunoglobulin inhibitor. In preferredembodiments the inhibitor(s) is/are IgA, IgM and IgG1. In someembodiments, the method also includes qualitatively and/orquantitatively testing a body fluid or secretion, such as saliva, forsaid at least one inhibitor to confirm immunization.

In accordance with still other embodiments of the invention, a method ofrestoring or enhancing immunoglobulin regulation of steroid hormoneresponsive cell growth in a mucosal epithelial cell is provided. Themethod comprises restoring or enhancing expression in the cell of amediator of immunoglobulin regulation chosen from the group consistingof a poly-Ig receptor and a Fcγ receptor. In some embodiments the methodcomprises inserting a gene for a poly-Ig receptor into said cell andexpressing said gene.

Also provided in accordance with certain embodiments of the presentinvention is a method of identifying carcinogenic bacteria. In certainembodiments the method includes: (a) obtaining a bacteria-containingspecimen of glandular/mucosal epithelial tissue or body fluid secretedby or bathing a gland or mucosal epithelial tissue; (b) takingprecautions in obtaining and handling said specimen such thatcontamination by extraneous microorganisms is avoided; (c) culturing thebacteria in said specimen such that at least one isolated bacterialcolony is obtained; (d) selecting at least one of said bacterialcolonies for further examination; and (e) conducting an Ames Test oneach selected colony such that mutagen-producing bacterial isolates areidentifiable. In certain embodiments the method also contains one ormore of the following steps: (f) determining the gram stain negative orgram stain positive classification of said bacterial colonies; (g)testing the bacterial isolates for production of defined metabolitesknown to or suspected of being mutagenic; (h) testing the bacterialisolates for induction of an oxidative burst when incubated with aneutrophil or macrophage; and (i) testing the bacterial isolates forimmunoglobulin protease activity. In some embodiments the method alsocontains one or more of the following steps: (j) when the fluidcomprises a breast secretion, determining whether a bacterial isolatesurvives and grows in the presence of a normal bacterial cell inhibitingamount of lactoferrin; (k) growing a bacterial isolate in a medium and,after growing the bacterial isolate, testing the medium with anon-tumorigenic human mucosal epithelial cell line such that cells thatare altered to a malignant phenotype by a component of the medium aredetectable; and (l) identifying a bacterial isolate using a polymerasechain reaction (PCR) technique.

In accordance with certain additional embodiments of the invention, amethod of conferring or enhancing resistance by a mucosal epithelialcell to malignant transformation is provided. The method comprisesinducing immunity in a host to at least one bacteria known to orsuspected of being oncogenic, as identified by the above-describedmethod.

In some embodiments of the invention a method of deterring malignanttransformation of a mucosal epithelial cell is provided that includesadministering an effective amount of an antibiotic to a host infected byan oncogenic bacteria, as identified by the above-described method.

In certain other embodiments, a method of suppressing an effect ofmalignant transformation of a mucosal epithelial cell is provided inwhich a cell growth arresting amount of at least one immunoglobulinchosen from the group consisting of dimeric or polymeric IgA, polymericIgM and IgG1 is administered to an individual in need thereof.

Still other embodiments of the present invention provide a method ofpreparing an anti-cancer antibody comprising selecting at least onebacteria known to, or suspected of, inducing malignant transformation inmucosal epithelial cells, according to the above-described method, andinducing immunity to the bacteria in an individual considered to be atrisk of developing cancer in a tissue comprising the cells.

Also provided in accordance with certain embodiments of the invention isa method of preventing or reducing the risk of developing cancer in amucosal epithelial tissue comprising immunizing an individual against atleast one bacteria known to or suspected of inducing malignanttransformation in that tissue. Preferably the bacteria is identified aspreviously described. In certain embodiments, the immunization comprisesorally, nasally or rectally administering an inactivated or attenuatedform of the bacteria to the individual such that mucosal immunityagainst the bacteria is conferred.

In certain embodiments of the invention, a method of suppressing aneffect of malignant transformation of a steroid hormone responsiveepithelial cell is provided. Representative steroid hormone responsiveepithelial cells are breast, prostate, oral cavity mucosa,salivary/parotid glands, esophagus, stomach, small intestine, colon,tear ducts, nasal passages, liver and bile ducts, bladder,secretory/exocrine pancreas, adrenals, kidney tubules, glomeruli, lungs,ovaries, fallopian tube, uterus, cervix, vagina, and secretory anteriorpituitary gland cells. The method comprises enhancing the amount of IgAand/or IgM and/or IgG1 secreted by or contacting the cell such thatsteroid responsive growth stimulation of the cell is inhibited in theabsence of a inhibition reversing amount of the steroid hormone or asteroid hormone mimicking substance.

In accordance with certain additional embodiments of the presentinvention, a method of detecting previous or active infection by abacteria known to or suspected of being oncogenic in mucosal epithelialtissue is provided which includes detecting in plasma or a body fluid orsecretion an antibody against the bacteria. In preferred embodiments thebacteria known to or suspected of being oncogenic is identified inaccordance with the above-described screening method.

In certain embodiments of the present invention, a method of preventingor reducing the risk of occurrence of cancer of a mucosal epithelialtissue, such as breast, prostate, colon, kidney and ovary, is provided.The method includes administering to a mammalian subject in need thereofat least one of the following treatments: (a) administering anantibiotic active against at least one bacteria known to or suspected ofinducing malignant transformation in mucosal epithelial cells; and (b)administering an immunogen to said subject in an amount sufficient toinduce plasma and/or mucosal production of at least one secretoryimmunoglobulin inhibitor of steroid hormone responsive cell growthsufficient to inhibit steroid hormone responsive proliferation of aplurality of steroid hormone responsive cancer cells in said mammal;administering at least one immunoglobulin inhibitor of steroid hormoneresponsive cell growth in an amount sufficient to inhibit or arreststeroid hormone responsive growth of said cells.

In accordance with certain other embodiments of the present invention, apharmaceutical composition is provided that comprises at least oneimmunoglobulin inhibitor of steroid hormone responsive cell growth and apharmacologically acceptable carrier. The cell is preferably a cancerousmucosal epithelial cell. In certain embodiments at least oneimmunoglobulin inhibitor is IgA, IgM or IgG1, preferably dimeric IgA,polymeric IgA, polymeric IgM or IgG1κ. In some embodiments thecomposition also contains one or more immunoglobulin inhibitor-mimickingsubstances, such as tamoxifen or a metabolite of tamoxifen.

Also provided in accordance with certain embodiments of the presentinvention is an anti-cancer composition comprising a pharmacologicallyacceptable carrier and a cytotoxic agent or a chemotherapeutic agentconjugated to an immunoglobulin inhibitor of steroid hormone responsivecancer cell growth. In preferred embodiments the inhibitor is IgA, IgM,IgG1, or any combination of those.

In accordance with certain other embodiments of the present invention amediator of steroid hormone reversible IgA and/or IgM inhibition ofsteroid hormone responsive cell growth is provided that comprises apoly-Ig receptor. In certain embodiments a mediator of steroid hormonereversible IgG1 inhibition of steroid hormone responsive cell growth isprovided which comprises a Fcγ receptor.

Also provided in certain embodiments of the present invention is anexpression vector for gene replacement therapy in a mammalian cell torestore or enhance expression of a poly-IgR. In some embodiments thevector comprises a preselected deoxyribonucleic acid (DNA) sequenceencoding a poly-Ig receptor, or a biologically active subunit or variantthereof, operably linked to a promoter capable of functioning in apreselected mammalian target cell. In some embodiments, an expressionvector for gene replacement therapy in a mammalian cell to restore orenhance expression of a Fcγ receptor is provided which comprises apreselected DNA sequence encoding a Fcγ receptor, or a biologicallyactive subunit or variant thereof, which is operably linked to apromoter functional in a preselected mammalian target cell.

Still other embodiments of the present invention provide an expressionvector for gene replacement therapy in a mammalian cell to restore orenhance expression of a TGFβ receptor. This vector comprises apreselected DNA sequence encoding a TGFβ receptor, or a biologicallyactive subunit or variant thereof, which is operably linked to apromoter functional in a preselected mammalian target cell.

Also provided in accordance with certain embodiments of the presentinvention is a method of expressing a DNA sequence encoding a mediatorof immunoglobulin inhibition of cell growth, the mediator being chosenfrom among a poly-Ig receptor, a Fcγ receptor, and biologically activesubunits and variants thereof operably linked to a promoter that iscapable of functioning in a preselected mammalian target cell. Themethod includes introducing the DNA sequence and the linked promoterinto the mammalian cell and allowing the cell to express the DNAsequence. In certain embodiments the also includes expressing a DNAsequence encoding a TGFβ receptor, or a biologically active subunit orvariant thereof, which is operably linked to a promoter that is capableof functioning in a preselected mammalian target cell. In thisembodiment the TGFβ receptor DNA sequence and its linked promoter areintroduced into the mammalian cell and the cell is allowed to expressthe DNA sequence.

These and other embodiments, features and advantages of the presentinvention will become apparent with reference to the followingdescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

For the detailed descriptions of the preferred embodiments, referencewill now be made to the accompanying figures which include graphs,charts, and test results:

FIG. 1. CDE-horse Serum Effect on MTW9/PL2 Cell Growth±10 nM E₂ for 7days. (A) Dose-response data expressed as cell numbers; (B)Dose-response data expressed as cell population doublings (CPD) per 7days.

FIG. 2. Restoration of Growth by Addition of 10 nM E₂ on days 0, 2, 4and 6 After Seeding the MTW9/PL2 cells into Inhibitory Medium Containing50% (v/v) of CDE-horse serum.

FIG. 3. Dose-Response Effects of Steroid Hormones on Growth of theMTW9/PL2 Cells in Medium Containing 50% (v/v) CDE-horse Serum.

FIG. 4. MTW9/PL2 Cell Growth±E₂ in Medium with CDE Sera from SeveralSpecies. (A) CDE-porcine Serum; (B) CDE-pregnant Human Serum; (C)CDE-adult Rat Serum; (D) CDE-adult Bovine Serum; (E) CDE-fetal BovineSerum; (F) CDE-fetal Horse Serum.

FIG. 5. CDE-horse Serum Effect on GH₄C₁ Cell Growth±10 nM E₂ for 10days.

FIG. 6. CDE-horse Serum Effect on ZR-75-1 Cell Growth±10 nM E₂ for 14days.

FIG. 7. CDE-horse Serum Effect on MCF-7A Cell Growth±10 nM E₂ for 10days.

FIG. 8. Kinetics of T47D Cell Growth in CDE-horse Serum±10 nM E₂. (A)Growth Kinetics in 20% CDE-horse±E₂ versus 10% Fetal Bovine Serum; (B)Growth Kinetics in 50% CDE-horse Serum±E₂.

FIG. 9. Rodent and Human ER⁺ Cell Line Growth in 50% CDE-human Serum±E₂.(A) T47D Human Breast Cancer Cells; (B) LNCaP Human Prostate CancerCells; (C) MTW9/PL2 Rat Mammary Tumor Cells; (D) GH₃ Rat Pituitary TumorCells; (E) GH₄C₁ Rat Pituitary Tumor Cells (F) H301 Syrian HamsterKidney Tumor Cells.

FIG. 10. Dose-Response of Steroid Hormones with T47D Cells in 50%CDE-horse Serum.

FIG. 11. Dose-Response of Steroid Hormones with GH₄C₁ Cells in 50%CDE-horse Serum.

FIG. 12. Dose-Response of Steroid Hormones with H301 Cells in 50%CDE-horse Serum.

FIG. 13. Dose-Response of Steroid Hormones with LNCaP Cells in 50%CDE-horse Serum.

FIG. 14. T₃ Growth Effects with GH₃ Cells in Serum-free Medium (PCM).

FIG. 15. E₂ Growth Effects with GH₃ Cells in Serum-free Medium (PCM)Minus E₂.

FIG. 16. T₃ Growth Effects with Three GH Cell Lines in 2.5% CDE-horseSerum.

FIG. 17. T₃ Growth Effects with Two GH Cell Lines in 50% CDE-horseSerum.

FIG. 18. Effect of XAD-4™ Resin Treated Horse Serum on MTW9/PL2 CellGrowth±E₂.

FIG. 19. Effect of XAD-4™ Resin Treated Horse Serum on T47D CellGrowth±E₂.

FIG. 20. Effect of Phenol Red on Estrogen Responsive MCF-7 Cell Growth.(A) MCF-7A Cell Growth in CDE-horse Serum±Phenol Red and ±E₂; (B)Estrogenic Effects with MCF-7A Cells±Phenol Red; (C) MCF-7K Cell Growthin CDE-horse Serum±Phenol Red and ±E₂; (D) Estrogenic Effects withMCF-7K Cells±Phenol Red.

FIG. 21. Effect of Phenol Red on Estrogen Responsive T47D and ZR-75-1Cell Growth; (A) T47D Cell Growth in CDE-horse Serum±Phenol Red and ±E₂;(B) Estrogenic Effects with T47D Cells±Phenol Red; (C) ZR-75-1 CellGrowth in CDE-horse Serum±Phenol Red and ±E₂; (D) Estrogenic Effectswith ZR-75-1 Cells±Phenol Red.

FIG. 22. Effect of Phenol Red on Estrogen Responsive MTW9/PL2 CellGrowth; (A) MTW9/PL2 Cell Growth in CDE-horse Serum±Phenol Red and ±E₂;(B) Estrogenic Effects with MTW9/PL2 Cells±Phenol Red.

FIG. 23. Dose-Response Effects of Phenol Red versus E₂ with Three ER⁺Cell Lines. (A) Growth Effects of Phenol Red with MCF-7K, T47D andMTW9/PL2 Cells; (B) Growth Effects of E₂ with MCF-7K, T47D and MTW9/PL2Cells.

FIG. 24. Estrogen Induction of Progesterone Receptors by Phenol Redversus E₂. (A) Induction by E₂ with T47D Cells; (B) Induction by PhenolRed with T47D Cells.

FIG. 25. Effects of TGFβ1 on Cell Growth in 2.5% CDE-horse Serum±E₂. (A)MCF-7K Cell Growth; (B) MTW9/PL2 Cell Growth.

FIG. 26. TGFβ1 Inhibition of ER⁺Rodent and Human Cell Line Growth±E₂.(A) Inhibition Data±E₂ Presented in Cell Number; (B) Inhibition Data±E₂Presented in CPD.

FIG. 27. EGF and TGFα as Substitutes for the Effects of E₂ in CDE-horseSerum. (A) MCF-7A Cell Growth; (B) MCF-7K Cell Growth; (C) T47D CellGrowth; (D) ZR-75-1 Cell Growth.

FIG. 28. IGF-I as a Substitute for the Effects of E₂ in CDE-horse Serum.(A) MCF-7K Cell Growth; (B) MCF-7A Cell Growth; (C) T47D Cell Growth.

FIG. 29. Growth of T47D Human Breast Cancer Cells in Standard and“low-Fe” D-MEM/F-12.

FIG. 30. Growth of LNCaP Human Prostate Cancer Cells in Standard and“low-Fe” D-MEM/F-12.

FIG. 31. Growth of MDCK Dog Kidney Tubule Cells in Standard and “low-Fe”D-MEM/F-12.

FIG. 32. Growth of AR⁺ LNCaP Cells in CAPM±DHT versus Growth inD-MEM/F-12 Containing 10% Fetal Bovine Serum.

FIG. 33 Growth of the AR⁻ DU145 and AR⁻ PC3 Cells in CAPM versus Growthin D-MEM/F-12 Containing 10% Fetal Bovine Serum.

FIG. 34. Dose-Response Effects of Individual Components of CAPMSerum-free Defined Medium on LNCaP Cell Growth.

FIG. 35. Effects of Deletion of Individual Components from CAPMSerum-free Medium on LNCaP, DU145 and PC3 Cell Growth±DHT.

FIG. 36. Effect of Fe (III) on MCF-7A Cell Growth in DDM-2MF Serum-freeDefined Medium.

FIG. 37. Effect of Fe (III) on T47D Cell Growth in DDM-2MF Serum-freeDefined Medium.

FIG. 38. Effect of Fe (III) on LNCaP Cell Growth in CAPM PlusApotransferrin.

FIG. 39. Comparative Effect of Fe (III) on LNCaP, DU145 and PC3 CellGrowth in CAPM.

FIG. 40. Growth Restoring Effect of Fe (III) Chelators in serum-freemedium with T47D Cells.

FIG. 41. Growth Restoring Effect of Fe (III) Chelators in serum-freemedium with LNCaP Cells.

FIG. 42. Comparison of DU145 Cell Growth in “low-Fe” and “standard”D-MEM/F-12 Based Serum-free Defined Medium CAPM.

FIG. 43. Comparison of PC3 Cell Growth in “low-Fe” and “standard”D-MEM/F-12 Based Serum-free Defined Medium CAPM.

FIG. 44. Growth of the DU145 Cells in CDE-horse Serum±DHT.

FIG. 45. Growth of the PC3 Cells in CDE-horse Serum±DHT.

FIG. 46. Growth of the ALVA-41 Cells in CDE-horse Serum±DHT.

FIG. 47. Comparison of Estrogenic Effects in Serum-free Defined Mediumand in D-MEM/F-12 Medium Supplemented with CDE-Horse Serum; (A) MCF-7KCell Growth in Serum-free Defined Medium±E₂; (B) MCF-7K Cell Growth inD-MEM/F-12 with CDE-horse Serum±E₂; (C) T47D Cell Growth in Serum-freeDefined Medium±E₂; (D) T47D Cell Growth in D-MEM/F-12 with CDE-horseSerum±E₂; (E) LNCaP Cell Growth in Serum-free Defined Medium±E₂; (F)LNCaP Cell Growth in D-MEM/F-12 with CDE-horse Serum±E₂.

FIG. 48. Effect of CDE-horse Serum on LNCaP Cell Growth in Serum-freeCAPM±E₂ and ±DHT.

FIG. 49. Comparison of Estrogenic Effects in Serum-free Defined Medium.

and in D-MEM/F-12 Medium Supplemented with CDE-Horse Serum.

GH₄C₁ Cell Growth in Serum-free Defined Medium±E₂.

GH₄C₁ Cell Growth in D-MEM/F-12 with CDE-horse Serum±E₂.

MTW9/PL2 Cell Growth in Serum-free Defined Medium±E₂.

MTW9/PL2 Cell Growth in D-MEM/F-12 with CDE-horse Serum±E₂.

H301 Cell Growth in Serum-free Defined Medium±E₂.

H301 Cell Growth in D-MEM/F-12 with CDE-horse Serum±E₂.

FIG. 50. Comparison of the Inhibitor Reversing Effects of DHT, E₂, andDES on LNCaP. Cell Growth in CDE-horse Serum Containing Medium; (A)Effect of DHT as an Inhibitor Reversing Steroid; (B) Effect of E₂ as anInhibitor Reversing Steroid; (C) Effect of DES as an Inhibitor ReversingSteroid; (D) Effect of Combinations of DHT, E₂, and DES as InhibitorReversing Steroids.

FIG. 51. Column Elution Profiles of the Two-step Cortisol Affinity andphenyl Sepharose Elution of CA-PA-pool I and CA-PS-pool II.

FIG. 52. Identification of the Molecular Forms Present in ActiveCA-PS-pool II. (A) SDS-PAGE with Coomassie Blue Staining; (B) WesternAnalysis with Anti-human SHBG.

FIG. 53. CA-PS-pool II Effect on ER⁺ Cell Growth in 2.5% CDE-horseSerum±E₂. (A) GH₁ Cells; (B) GH₃ Cells; (C) GH₄C₁ Cells; (D) H301 Cells;(E) MTW9/PL2 Cells; (F) MCF-7K Cells; (G) ZR-75-1 Cells (II) T47D Cells.

FIG. 54. Cortisol Affinity Column Depletion of the Estrogenic Activityin CDE-horse Serum Assayed with ER⁺ Cell Lines±E₂. (A) T47D CellsPre-Column; (B) T47D Cells Post-Column; (C) GH₃ Cells Pre-Column; (D)GH₃ Cells Pre-Column; (E) H301 Cells Pre-Column; (F) H301 CellsPost-Column.

FIG. 55. Serum-free Growth of Cells in Four Different Defined Media±E₂.(A) MTW9/PL2 Cells in DDM-2A; (B) T47D Cells in DDM-2MF; (C) GH₄C₁ Cellsin PCM-9; and (D) H301 Cells in CAPM.

FIG. 56. Effects of CDE-horse Serum on Estrogen Responsiveness of ThreeER⁺ Cell Lines Growing in Serum-free Defined Media. (A) T47D Cells inDDM-2MF; (B) MTW9/PL2 Cells in DDM-2A; (C) GH₄C₁ Cells in PCM-9.

FIG. 57. Effects of CA-PS-pool II on the Growth of Eight ER⁺ Cell Linesin Serum-free Defined Medium±E₂.

FIG. 58. Western Analysis of CA-PS-pool I and CA-PS-pool II with theAntibody Raised to the 54 kDa Band.

FIG. 59. Effect of the Anti-54 kDa Antiserum on the Inhibition ofMWT9/PL2 Cell Growth by the Isolated Fraction CS-PS-Pool II.

FIG. 60. Western Immunoblotting of Commercially Prepared Horse IgG, IgAand IgM with anti-54 kDa Antiserum.

FIG. 61. Effect of Horse IgG on MTW9/PL2 Cell Growth in 2.5% CDE-horseSerum±E₂.

FIG. 62. Effect of Horse IgM on MTW9/PL2 Cell Growth in 2.5% CDE-horseSerum±E₂.

FIG. 63. Effect of Horse IgA on MTW9/PL2 Cell Growth in 2.5% CDE-horseSerum±E₂.

FIG. 64. SDS-PAGE with Coomassie Staining and Western Analysis of RatPurified “SHBG-like” Proteins. (A) SDS-PAGE of Purified RatPreparations; (B) Western Analysis with Anti-rat IgG.

FIG. 65. Western Analysis of a Rat Purified “SHBG-like” Preparation. (A)Western with Anti-rat IgA with Purified IgA Control; (B) Western withAnti-rat IgG1 with Purified IgG1 Control; (C) Western with Anti-rat IgMwith Purified IgM Control.

FIG. 66. Comparison of Rat IgG Subclasses for Antibody Cross-Reaction.(A) SDS-PAGE with Coomassie Blue Staining; (B) Western Analysis withRabbit Anti-Human SHBG.

FIG. 67. Effect of Rat IgG on MTW9/PL2 Cell Growth in Medium with 2.5%CDE-rat Serum±E₂.

FIG. 68. Effect of Rat IgA on MTW9/PL2 Cell Growth in Medium with 2.5%CDE-rat Serum±E₂.

FIG. 69. Effect of Rat IgM on MTW9/PL2 Cell Growth in Medium with 2.5%CDE-rat Serum±E₂.

FIG. 70. Mannan Binding Protein Isolation of Human Plasma/Serum IgM.

FIG. 71. Jacalin Lectin Purification of Human Plasma/Serum IgA.

FIG. 72. Effect of Human IgM on MTW9/PL2 Cell Growth±E₂ in Serum-freeDefined Medium.

FIG. 73. Comparison of the Effects of Rat and Horse IgA and IgM onMTW9/PL2 Cell Growth±E₂ in Serum-free Defined Medium Expressed in CellNumber and CPD.

FIG. 74. Effect of Rat Myeloma IgA on GH₁ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 75. Effect of Human Plasma IgA on GH₁ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 76. Effect of Human Plasma IgM on GH₁ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 77. Effects of sIgA on GH₁ Cell Growth in Serum-free DefinedMedium±E₂.

FIG. 78. Model of Mucosal Epithelial Cell Transport of IgA/IgM.

FIG. 79. Essential Structures of Human Plasma and Secretory IgA.

FIG. 80. Effect of Rat Myeloma IgA on GH₃ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 81. Effect of Rat IgM on GH₃ Cell Growth in Serum-free DefinedMedium±E₂.

FIG. 82. Effect of Human Plasma IgA on GH₃ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 83. Effect of Human Plasma IgM on GH₃ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 84. Effect of Human Secretory IgA on GH₃ Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 85. Effect of Rat Myeloma IgA on GH₄C₁Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 86. Effect of Rat Plasma IgM on GH₄C₁Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 87. Effect of Human Plasma IgA on GH₄C₁Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 88. Effect of Human Plasma IgM on GH₄C₁Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 89. Effect of Human Secretory IgA on GH₄C₁Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 90. Effect of Mouse IgA on H301 Cell Growth in Serum-free DefinedMedium±E2.

FIG. 91. Effect of Human IgA on H301 Cell Growth in Serum-free DefinedMedium±E2. (A) Plasma IgA Effects; (B) Secretory sIgA Effects.

FIG. 92. Dose-Response Effects of E₂ on H301 Cell Growth in Serum-freeDefined Medium Containing 40 μg/mL Human Plasma IgM.

FIG. 93. Effect of Human IgA on MCF-7A Cell Growth in Serum-free DefinedMedium±E2. (A) Plasma IgA Effects; (B) Secretory sIgA Effects.

FIG. 94. Effect of Human IgA on MCF-7K Cell Growth in Serum-free DefinedMedium±E2. (A) Plasma IgA Effects; (B) Secretory sIgA Effects.

FIG. 95. Effect of Human IgM on MCF-7A Cell Growth in Serum-free DefinedMedium±E₂.

FIG. 96. Effect of Human IgM on MCF-7K Cell Growth in Serum-free DefinedMedium±E₂.

FIG. 97. Dose-Response Effects of E₂ on MCF-7K Cell Growth in Serum-freeDefined Medium Containing 40 μg/mL Human Plasma Ig.

FIG. 98. Effect of Human IgA on T47D Cell Growth in Serum-free DefinedMedium±E2. (A) Plasma IgA Effects; (B) Secretory sIgA Effects.

FIG. 99. Effect of Human IgM on T47D Cell Growth in Serum-free DefinedMedium E₂.

FIG. 100. Dose-Response Effects of E₂ on T47D Cell Growth in Serum-freeDefined Medium Containing 40 μg/mL Human Plasma IgM.

FIG. 101. Effect of Human. IgA on ZR-75-1 Cell Growth in Serum-freeDefined Medium±E2. (A) Plasma IgA Effects; (B) Secretory sIgA Effects.

FIG. 102. Effect of Human IgM on ZR-75-1 Cell Growth in Serum-freeDefined Medium±E₂.

FIG. 103. Effect of Human IgM on HT-29 Cell Growth in Serum-free DefinedMedium±T3.

FIG. 104. Effect of Human IgA on LNCaP Cell Growth in Serum-free DefinedMedium±E2. (A) Plasma IgA Effects; (B) Secretory sIgA Effects.

FIG. 105. Effects of Human Plasma versus Human Myeloma IgM on LNCaP CellGrowth in Serum-free Defined Medium±DHT.

FIG. 106. Summary of Estrogenic Effects with Various ER⁺ Cell lines andDifferent Ig Sources.

FIG. 107. Effect of Tamoxifen on T47D Cell Growth in Serum-free DefinedMedium.

FIG. 108. Estrogen Reversal of Tamoxifen Inhibition of T47D cells inSerum-free Defined Medium.

FIG. 109. Estrogen Rescue of MTW9/PL2 Cell Growth in Serum-free MediumContaining 40 μg/mL of Horse Serum IgM.

FIG. 110. Summary of Estrogen Rescue of MTW9/PL2 Cell Growth inSerum-free Medium Containing 40 μg/mL of Horse Serum IgM.

FIG. 111. Estrogen Rescue of T47D Cell Growth in Serum-free MediumContaining 40 μg/mL of Human Serum IgM.

FIG. 112. Estrogen Rescue of MCF-7A Cell Growth in Serum-free MediumContaining 40 μg/mL of Human Serum IgM.

FIG. 113. Western Detection of the Secretory Component of Human MilksIgA.

FIG. 114. Effect of Anti-Secretory Component on IgM Inhibition of T47DCell Growth in Serum-free Defined Medium.

FIG. 115. Effect of Anti-Secretory Component on pIgA Inhibition of LNCaPCell Growth in Serum-free Defined Medium.

FIG. 116. Western Analysis with Anti-Secretory Component to Detect thePoly-Ig Receptor in AR⁺ and AR⁻ Prostate Cancer Cells plus Control CellLines.

FIG. 117. Effect of Human pIgA on DU145 Cell Growth in Serum-freeDefined Medium±DHT.

FIG. 118. Effect of Human pIgA on PC3 Cell Growth in Serum-free DefinedMedium±DHT.

FIG. 119. Effect of Rat Immunoglobulins on Estrogen Responsive Growth ofMTW9/PL2 Cells In Serum-free Defined Medium.

FIG. 120. Comparison of the Estrogenic Effects of Human Immungobulinwith T47D Cells in Serum-free Defined Medium.

FIG. 121. Effect of Human IgG Isotypes on LNCaP Cell Growth inSerum-free Defined Medium±DHT.

FIG. 122. IgG Isotype Assays with LNCaP Cells in Serum-free DefinedMedium±DHT.

FIG. 123. Model of Early Onset Breast Cancer Including TGFβ.

FIG. 124. Effect of Carcinogens on Mammary Tumor Induction in Rats ofVarious Ages.

FIG. 125. Anti-human SHBG Antibody Immunoprecipitation of the EstrogenicActivity Present in CDE-horse Serum Assayed with MTW9/PL2 Cells.

FIG. 126. Anti-human SHBG Antibody Immunoprecipitation of the EstrogenicActivity Present in CDE-rat Serum Assayed with MTW9/PL2 Cells.

FIG. 127. Anti-human SHBG Antibody Immunoprecipitation of the LabeledSteroid Hormone Binding Activity Present in CDE-rat Serum.

FIG. 128. Western Analysis and Densitometry of the Immunoglobulin Levelsin the Serum of Female Rats of Specified Age Groups.

FIG. 129. Structural and Functional Organization of the Human EstrogenReceptor α.

FIG. 130. Entre Genome NCBI Search of “Breast Cancer Mutations” andChromosomes.

FIG. 131. Chromosome 0.1 Map of Breast Cancer Loci versus the Poly-IgReceptor Locus.

FIG. 132. Colon, Breast and Prostate Cancer Death Rates Around theWorld.

FIG. 133. Immunoglobulin IgG, IgA and IgM Concentrations in Plasmaversus Human Age.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

To facilitate review of the detailed description of preferredembodiments, a Table of Contents is provided. The titles used for thevarious subsections and examples are not intended to be limiting and areonly an aid to locating certain subject matter. In addition, some of theExamples that follow begin with a short introduction and/or summary,which is intended merely to facilitate review and is not limiting on thedisclosure contained in the full Example, and end with a Discussion,which may include some conclusions that may be drawn from that Example.

Table of Contents Subsection Paragraph No. Introduction 199 Example 1:Methods and Compositions For Demonstration of Steroid 206 HormoneResponsive Cancer Cell Growth in Culture Example 2: Preparations ofSteroid Hormone Depleted Serum 224 Example 3: MTW9/PL2 Rat Mammary TumorCell Estrogen Responsive Growth in 34° C. 230 Charcoal-dextran ExtractedSerum Example 4: Estrogen Responsive Growth of Additional Rodent andHuman Cell Lines In 34° C. 245 Charcoal-dextran Extracted Horse andHuman Serum Example 5: Thyroid Hormone Growth Effects in CDE-Horse SerumPrepared at 34° C. 257 Example 6: Estrogenic Effects in XAD-4 ™ ResinTreated Horse Serum 259 Example 7: Testing of Substances for EstrogenicActivity 261 Example 8: Roles of TGFβ and Growth Factors: ConceptualImplications 275 Example 9: Serum-free Defined Culture MediumCompositions 293 Example 10: Serum-free Defined Medium Supports BothHormone Sensitive 316 and Autonomous Cancer Cells Example 11:Differential Effects of Fe (III) on the Growth of Hormone Responsive and322 Autonomous Human Breast and Human Prostate Cancer Cells Example 12:Growth in Serum-free Defined Medium versus Growth in CDE-Serum ± E₂ 332Example 13: Action of DES on Human AR ⁺LNCaP Prostate Cancer Cells 341Example 14: Properties and Rationale For Serum Purification Source 343Example 15: Cortisol Affinity and Phenyl Sepharose Isolation of the“SHBG-like” 347 Estrogen Reversible Inhibitor from CDE-Horse SerumExample 16: Serum-free Assay Systems for Measuring Large MagnitudeSteroid Hormone 366 Mitogenic Responses with the Two-Step PurifiedInhibitor Example 17: Chemical and Immunological Properties of thePartially Purified CA-PS-Pool II 370 Inhibitors and Identification asIgA and IgM Example 18: Regulation of Steroid Hormone-responsive andThyroid Hormone-responsive 391 Cancer Cell Growth in Serum-free DefinedMedium by Secretory and Plasma Forms of IgA and Plasma and Cell CultureDerived IgM Example 19: A New High Estrogen Affinity Growth RegulatingEstrogen Receptor (ERγ) 407 Example 20: Effect of Tamoxifen Antiestrogenin Serum-free Defined Medium 419 Example 21: Effect of Long-TermExposure of Breast Cancer Cells to IgM Under 428 Serum-free DefinedConditions Example 22: The Role of the Poly-Ig Receptor in HormoneResponsive and 433 Autonomous Breast and Prostate Cell Growth RegulationExample 23: IgG1 and IgG2 as Immunoglobulin Regulators of Estrogen and445 Androgen Responsive Cancer Cell Growth Example 24: Mediation ofIgG1κ Effects by a Fc-like Receptor 452 Example 25. ImmunoglobulinInhibitors as Tools for Identifying the Receptors that 455 Mediate theIgA/IgM/IgG Cell Growth Regulating Effects Example 26: Conceptual Modelfor Cascading Loss of Immunoglobulin Control in Progression 466 fromNormal Cells to Steroid Hormone Responsive and Autonomous CancersExample 27: Role of TGFβ in Breast Cancer Predicts the CellularProgression 475 in Early Onset Breast Cancer Example 28: Windows ofBreast Susceptibility to Carcinogenesis and Mutation 480 and the Levelsof Immunoglobulin Inhibitors Example 29: Risk Factors: IgA/IgM BasedTest to Detect Lowered Levels of Steroid 492 Hormone Reversible CellGrowth Inhibitors in Plasma or Body Secretions Example 30: Risk Factors:IgA Deficiencies and Malignancies 498 Example 31: Risk Factors:Autoimmunity Test for Anti-IgA and IgM In Plasma 500 Example 32:Diagnostic and Prognostic Tools: Estrogen Receptor γ (ERγ) 505 Example33: Diagnostic, Prognostic and Treatment Decision Tools: Poly-IgReceptor 511 (or Poly-Ig like Receptor) Example 34: Diagnostic Tools:Monoclonal Antibodies to the Poly-Ig Receptor 517 and Breast CancerImaging Example 35: Diagnostic, Prognostic and Treatment Decision Tools:Fc-like 520 Receptor for IgG1/IgG2 Example 36: Diagnostic, Prognosticand Treatment Decision Tools: TGFβ Receptors 524 Example 37: AtaxiaTelangiectasia as an Example of a Human Genetic Disorder 529 with HighRates of Breast Cancer Coupled with an IgA Deficiency Example 38:Diagnostic and Predictive: Poly-Ig Receptor Based Genetic Screening 531for Breast, Prostate and other Mucosal Cancer Susceptibility Example 39:Treatment: Breast Cancer Prevention with Applications to Prostate Cancer543 and other Mucosal Cancers Example 40: Treatment: Rat Model forTesting Oral Immunization Effects on 558 Mammary Gland CarcinogenesisExample 41: Treatment: Bacterial Oncogenesis and Prevention by OralImmunization 573 Example 42: Treatment: Treatment of Steroid HormoneResponsive Breast or Prostate Cancer 601 by Administration ofIgA/IgM/IgG1 Example 43: Treatment: Monoclonal Antibodies that Mimic orblock IgA or IgM Binding 609 to the Poly-Ig Receptor Example 44:Treatment: Delivery of Chemotherapeutic Agents and Cytotoxins to CancerCells 616 via IgA/IgM/IgG1 or Monoclonal Antibodies to Poly-Ig ReceptorIntroduction

In the course of searching for what causes the growth of estrogenresponsive breast and androgen responsive prostate cancers, it wasdiscovered that the secretory immune system plays a major role in thosediseases. More specifically, it was discovered that the secretory immunesystem (i.e., the immunoglobulins IgA, IgM and IgG1) provide negative(inhibitory) regulation of steroid hormone responsive mucosal epithelialcancer cell growth in serum-free model cell culture systems, includingbreast, prostate, pituitary, kidney, colon, and other glandular cancercells. Prior to that discovery, which is described in co-ownedconcurrently-filed U.S. patent application Ser. No. 09/852,958PCT/US2001/15183 entitled “Compositions and Methods for DemonstratingSecretory Immune System Regulation of Steroid Hormone Responsive CancerCell Growth,” hereby incorporated herein by reference, no cell growthregulating role was known for the secretory immune system. The secretoryimmune system produces predominantly dimeric/polymeric IgA, secretoryIgA (sIgA), polymeric IgM, and IgG1. The discovery of immunoglobulininhibitors of cell growth is a major breakthrough in the understandingof cancers of breast and prostate, as well as other glandular/mucosaltissues that secrete or are bathed by the secretory immunoglobulins.

For the first time, a direct link is established between the secretoryimmune system and the most prevalent types of cancer that occurthroughout the world. Binding of IgA and IgM to the polyimmunoglobulinreceptor (poly-Ig receptor) is an important step in carrying out theregulatory function of IgA and IgM, and it is probable that the knownpoly-Ig receptor, or a closely related poly-Ig like receptor, mediatesthe negative regulation of steroid hormone dependent cell growth.Similarly, it is believed that the binding of IgG1 to the Fcλ receptoris a mediating step in carrying out the regulatory function of IgG1. Theapplication of this new understanding of immune system regulation ofcancer cell growth to the risk assessment, detection, diagnosis,prognosis, treatment and deterrence or prevention of a host of mucosalepithelial cell cancers is described in the following examples.

A new conceptual model described herein, offers an explanation of hownormal breast tissue may give rise to highly malignant, and dangerous,hormone autonomous forms. This model is contrary in some respects to thewell-established “linear progression” model, in which breast cancerspass through a characteristic natural history that involves a gradualevolution from near normal growth patterns into cancers that arecompletely steroid hormone autonomous (i.e., they are no longerstimulated by steroid hormones), and describes cell growth regulatoryroles for TGFβ, IgA, IgM and IgG1.

The secretory immune system was not previously known to have any cellgrowth regulatory role in breast or prostate cancer, or in other cancersof the mucus epithelial tissues. As set forth in various of thefollowing examples, new compositions and “immunotherapy” protocols basedon production or administration of IgA, IgM and IgG1, as well as newmethods of immune-related diagnosis and assessment of susceptibility areprovided. Also, effective new gene expression and gene transfectiontherapies by which malignant breast cells may be returned to naturalimmune control are described. Such strategies will be far less toxicthan those now employing chemotherapeutic agents. A significant featureof the discovery is that a new natural “immune” mechanism exists that isdistinct from the anti-tumor immunological approaches of the past, andwhich can be exploited to control breast cancer.

It should be readily appreciated that this discovery has implicationswell beyond cancers of the breast and prostate. The secretory immunesystem is an integral part of the physiology of all mucosal epithelialtissues. Most, if not all, mucosal tissues secrete IgA, IgM and IgG1directly into the lumen of biological passageways. This includessalivary glands, oral and nasal cavities, stomach, small and largebowel, lung passageways, the kidney tubule, liver and bile ducts,prostate, bladder, the anterior pituitary, and the secretory/exocrinepancreas. Secretory immune system control of cell proliferation is alsorelevant to cancers of the female reproductive tract. The entire femalereproductive tract including ovaries, uterus, cervix and vagina eithersecretes IgA and IgM or is a target for these immunoglobulins. In fact,malignancies of all secretory epithelial tissues represent 80% or moreof the cancers in human females.

The compositions and methods, and the biochemical, genetic andimmunological tools described herein, and those described in U.S. patentapplication Ser. No. 09/852,958 PCT/US2001/15183 entitled “Compositionsand Methods for Demonstrating Secretory Immune System Regulation ofSteroid Hormone Responsive Cancer Cell Growth” (hereby incorporatedherein by reference), are employed in the present investigations tofurther elucidate the cascade of cellular changes that lead tomalignancy in glandular/mucosal tissues and to provide, among otherthings, ways of testing cancer cells for loss of IgA/IgM/IgG1regulation, ways to detect genetic changes in the poly-Ig receptor,biochemical and genetic screening procedures to identify individuals athigh risk for developing breast or prostate cancer, and ways ofdeterring or reducing the risk of development of such cancers.Additionally, in light of the discovery that the secretory immune systemimmunoglobulins IgA, IgM and IgG1 are potent inhibitors of steroidhormone responsive cancer cell growth, it is now proposed that thesteroid hormone responsive tissues in the body can be protected from thecancer causing actions of certain environmental carcinogens, especiallyduring age related “windows” of increased susceptibility, by enhancementof the IgA/IgM/IgG1 secreted by or coming in contact with those tissues.In this way, DNA synthesis dependent mutations can be prevented orsubstantially reduced in those tissues. Likewise, deleteriousdown-modulation or inactivation of critical gene expression (e.g., thepoly-Ig receptor) due to environmental carcinogens may also beremediable by restoration of IgA, IgM and/or IgG1 control of cellgrowth.

Also described in examples that follow is the use of cell growthinhibitory amounts iron, in the form of Fe (III) to treat malignanciesand/or surgical sites. Still other Examples which follow describescreening procedures for detecting potentially cancer-inducing bacteria,and offer preventative measures for decreasing the effects of bacterialcarcinogesis.

EXAMPLES Example 1 Methods and Compositions for Demonstration of SteroidHormone Dependent Cancer Cell Growth in Culture

In the following Examples, which describe representative, preferredembodiments of the present invention, the following general materialsand methods are employed, except as otherwise noted therein.

Cell Culture Medium.

The water used to prepare culture media and all other solutions waspurified first by reverse osmosis followed by passage through a U.S.Filter Corporation system with a charcoal filter and two mixed bed ionexchangers. The effluent was distilled using a Bellco glass apparatuswith quartz heating elements. The distilled water was stored in airflowrestricted glass containers. No metal fittings are allowed in contactwith the final purified water. This necessary precaution minimizesrecontamination with metal ions. Standard phenol red containing Ham'sF12-Dulbecco's modified Eagle's medium (D-MEM/F-12), phenol red-freestandard D-MEM/F-12 and a custom-prepared “low-Fe” D-MEM/F-12 mediumwere supplied by Gibco-BRL (Catalog No. 11330-032) or Bio♦Whittacker(Catalog No. 12-719, liquid). The “low-Fe” medium was standard phenolred containing D-MEM/F-12 from which the usual additions of ferricnitrate and ferrous sulfate had been omitted (Eby J E et al. (1992) AnalBiochem 203, 317-325; Eby J E et al. (1993) J Cell Physiol 156,588-600). This medium was a special formulation purchased from Gibco-BRLas a powder and prepared in the highly purified water before 0.2 μm porefilter membrane sterilization. A number of other stock solutions arerequired for cell culture in either serum containing or serum-freedefined medium. Descriptions of each preparation are provided along withspecific instructions for their use. The solutions used were designed tominimize the exogenous content of steroid hormone and to minimize the Fe(III) content of the water. Steps are taken for the exclusion of allextraneous sources of steroid hormones and Fe (III). Exclusion of Fe(III) is highly preferred, and in most of the totally serum-freeapplications, it is considered essential. Wherever possible, disposableplastic ware or glassware is used to minimize potential contamination.It is important to note that excess solutions are preferably discardedafter use with each individual cell line to avoid cross-contamination ofcell types (Nelson-Rees W A and Fladermeyer R R (1977) Science (Wash DC)195, 134-136).

General Cell Culture—Serum.

Adult and fetal horse, adult pig, adult sheep and adult and fetal bovineserum were obtained from Gibco-BRL. A mixture of adult male and femalerat serum was purchased from Pel-Freez, Rodgers, Ark. Human serum waspurchased from Bio♦Whittacker. Human plasma was a pool of samplescollected from pregnant females during routine visits to a local clinic.All serum was stored frozen at −20° C. until used. Repeated freeze-thawof serum or plasma is avoided. Before charcoal extraction, the EDTA wasremoved by dialysis at 7° C. for 24 hours against forty volumes of 0.05M Tris-HCl, pH 7.4, containing 50 mM CaCl₂. Dialysis was done withSpectropor 1 membranes (Spectrum Medical Industries, molecular weightcut-off 6,000 to 8,000). The clotted material was removed bycentrifugation. This preparation is termed plasma-derived serum. Theserum or plasma was not heat pre-treated, or heat inactivated prior touse in the methods described below.

General Cell Culture—Normal Saline.

Sterile normal saline (0.15 M NaCl) was prepared in 10 mL aliquots andstored at room temperature. Unused portions are discarded at the end ofeach experiment. A large supply is sterilized by autoclaving and used toprepare the solutions described below.

General Cell Culture—Trypsin/EDTA for Subculture.

Sterile preparations were purchased from Irvine Scientific (Catalog No.9341) or Bio♦Whittacker (Trypsin-Versene EDTA Mixture) (Catalog No.17-161F). This preparation contained 0.5 g/L trypsin and 0.2 g/L EDTA inHank's balanced salts solutions with 10 mg/L phenol red. Thispreparation does not contain Ca or Mg salts nor does it have NaHCO₃. Totrypsinize cells, 1.5 mL of this preparation was typically used.Aliquots (2 mL) were stored frozen until used and residual solutiondiscarded at the end of each experiment or application to a cell line.

General Cell Culture—Soybean Trypsin Inhibitor (STI).

STI was purchased from Sigma (Catalog No. T9128, Type II-2). An amountof 1.0 mg of this preparation will inactivate 1.0 mg of trypsinactivity. The solution is prepared as 0.2% (w/v) in normal saline andsterilized using a 0.2 μm pore diameter filter membranes. Aliquots of3.0 mL are stored at −20° C. until used. This preparation is used tostop the action of trypsin during harvest of stock cultures for growthassays. STI ensures that all trypsin used to harvest cells for growthassays is inactivated and therefore will not damage the proteinadditions to serum-free defined medium. Also, use of STI ensures that noextraneous steroid hormones are introduced after harvest of cells fromthe stock culture dishes.

General Cell Culture—Crude Pancreatic Trypsin for Cell Counting.

This trypsin preparation was used to harvest the cells for determiningcell numbers. The cells are typically grown in 35-mm diameter dishes.This enzyme was purchased from ICN Biochemicals as the 1-300 porcinepancreatic trypsin preparation (Catalog No. 103140). A stock solution istypically prepared by adding the contents of a preweighed bottle of 1×Dulbecco's modified PBS medium without calcium or magnesium to 800 mL ofwater. This solution dissolves very gradually with adjustment to pH 7.3using NaOH. After the solution was clear, 20 g of crude trypsin wasadded and this mixture stirred for 30 minutes at room temperature. Thesomewhat cloudy solution was diluted to 1000 mL with water and thisvolume was stored frozen in bulk overnight at −20° C. to induce coldrelated precipitation that typically occurs when this preparation wasfrozen and thawed. After thawing at 37° C. in a water bath, thepreparation was filtered through 0.45 μm pore membranes. Thispreparation was stored at −20° C. in useable portions.

General Cell Culture—EDTA for Cell Counting.

The EDTA used is the disodium and dihydrate salt (Sigma Catalog No.E1644). A 0.29 M solution is prepared by adding 107.9 g to 800 mL ofwater with stirring and adjustment to pH 7.2 with NaOH. The volume isbrought to one liter with water and the solution stored at roomtemperature. Because this solution is used only at the end of theexperiments, it does not require sterilization.

General Cell Culture.

In Table 1 the cell lines used in the described Examples are listed. Theabbreviation “KCC” is the Karmanos Cancer Center, Cell Line Repository,Detroit, Mich. The abbreviation “ATCC” is the American Type CultureCollection, Cell Line Repository, Manassas, Va. Professor ArmenTashjian's address is Harvard University, Boston, Mass. Dr. WilliamRosner's address is Columbia University, New York. Dr. Sirbasku'saddress is The University of Texas, Houston, Tex. The superscriptdesignations in Table 1 for each of the cell lines indicate referencesthat verify that the estrogen and androgen responsive cell lines used inthis study are bona fide hormone responsive based on their tumor formingcharacteristics in host animals. Those reports are clear demonstrationsof the reliability of the models used in the present investigations tostudy sex hormone dependence in culture.

TABLE 1 Cell Lines Employed in the Examples. ER⁺indicates receptorcontaining/E₂ sensitive CELL LINES SOURCES REFERENCES/CELL LINE ORIGINMCF-7K¹ KCC Soule HD et al. (1973) J Natl Cancer Inst 51, 1409-1416 ER⁺human breast cancer MCF-7A¹ ATCC Soule HD et al. (1973) J Natl CancerInst 51, 1409-1416 ER⁺ human breast cancer T47D² ATCC Keydar I et al.(1979) Eur J Cancer 15, 659-670 ER⁺ human breast cancer ZR-75-1³ ATCCEngle LW et al. (1978) Cancer Res 38, 3352-3364. ER⁺ human breast cancerGH₄C₁ ⁴ Dr. A. Tashjian Tashjian AH Jr (1979) Methods Enzymol 58,527-535 ER⁺ rat pituitary tumor GH₃ ⁵ ATCC Tashjian AH Jr (1979) MethodsEnzymol 58, 527-535. ER⁺ rat pituitary tumor GH₁ ATCC Tashjian AH Jr(1979) Methods Enzymol 58, 527-535 ER⁺ rat pituitary tumor MTW9/PL2⁶ Dr.D. Sirbasku Danielpour D et al. (1988) In Vitro Cell Dev Biol 24, 42-52ER⁺ rat mammary tumor H301⁷ Dr. D. Sirbasku Sirbasku DA and Kirkland WL(1976) Endocrinology 98, 1260-1272 ER⁺ Syrian hamster kidney tumorLNCaP⁸ ATCC Horoszewicz JS et al. (1983) Cancer Res 43, 1809-1818 AR⁺human prostatic carcinoma Fibroblasts Dr. D. Sirbasku Primary culturesof human foreskin and rat ear cartilage; Eastment CT and Sirbasku DA(1980) In Vitro 16, 694-705 ALVA-41 Dr. W. Rosner Nakhla AM and Rosner W(1994) Steroids 59, 586-589 AR⁻ human prostate cancer; androgen growthinsensitive DU145 ATCC Stone KR et al. (1978) Int J Cancer 21, 274-281AR⁻ human prostate cancer; androgen growth insensitive PC3 ATCC KaighnME et al. (1979) Invest Urol 17, 16-23 AR⁻ human prostate cancer;androgen growth insensitive HT-29 ATCC Chen TR et al. (1987) CancerGenet Cytogenet 27, 125-134 Thyroid hormone responsive human coloncancer ER⁺ indicates estrogen receptor containing. AR⁺ indicatesandrogen receptor containing. Unless otherwise noted, these designationsindicate sex steroid hormone growth responsive in culture.

In Vivo Tumor Forming Properties.

The references below refer to the superscript designations in Table 1for the cell lines. The references verify that the estrogen and androgenresponsive cell lines used in this disclosure are hormone responsivebased on their tumor forming characteristics in host animals. Thesereports are demonstration the reliability of the models used in thisdisclosure to study sex hormone dependence in culture.

-   ¹The use of two strains of MCF-7 cells has been described (Sirbasku    D A and Moreno-Cuevas (2000) In Vitro Cell Dev Biol 36, 428-446).    Clonal variations of this line are known (Seibert K et al. (1983)    Cancer Res 43, 2223-2239). Demonstration of estrogen responsive    MCF-7 tumor formation in vivo (Huseby R A et al. (1984) Cancer Res    44, 2654-2659; Soule H D and McGrath C M (1980) Cancer Lett 10,    177-189; Welsch C W et al. (1981) Cancer Lett 14, 309-316).-   ²Estrogen responsive T47D tumors in vivo (Leung C K H and Shiu R P    C (1981) Cancer Res 41, 546-551).-   ³Estrogen responsive ZR-75-1 tumors in vivo (Osborne C K et    al. (1985) Cancer Res 45, 584-589).-   ⁴Estrogen responsive GH₄C₁ tumors in vivo (Riss T L and Sirbasku D    A (1989) In Vitro Cell Dev Biol 25, 136-142).-   ⁵Estrogen responsive GH₃ tumors in vivo (Sorrentino J M et    al. (1976) J Natl Cancer Inst 56, 1149-1154).-   ⁶Estrogen responsive MTW9/PL2 tumors in vivo (Sirbasku D A (1978)    Cancer Res 38, 1154-1165; Danielpour D and Sirbasku D A (1984) In    Vitro 20, 975-980).-   ⁷Estrogen responsive H301 tumors in vivo (Sirbasku D A and Kirkland    W L (1976) Endocrinology 98, 1260-1272; Liehr J G et al. (1986) J    Steroid Biochem 24, 353-356).-   ⁸Androgen responsive LNCaP tumors in vivo (Sato N et al. (1997)    Cancer Res 57, 1584-1589; Gleave M et al (1991) Cancer Res 51,    3753-3761; Horoszewicz J S et al. (1983) Cancer Res 43, 1809-1818;    Pretlow T G et al. (1991) Cancer Res 51, 3814-3817; Passaniti A et    al. (1992) Int J Cancer 51, 318-324).

General Cell Culture—Cell Passage Method.

All stock cultures were grown in medium containing phenol red. Stocks ofthe cells were maintained at 37° C. in a humid atmosphere of 5% (v/v)CO₂ and 95% (v/v) air in 17 to 20 mL of standard D-MEM/F-12 with 2.2 gper liter sodium bicarbonate, 15 mM HEPES (pH 7.4), and serum. With allcell lines except the rat pituitary cells, the serum used for stockculture was 10% (v/v) fetal bovine serum (FBS). For the three ratpituitary tumor cell lines GH₄C₁, GH₁ and GH₃, the medium contained12.5% (v/v) horse serum and 2.5% (v/v) FBS. To passage the cells, themedium was removed and the dishes washed with 10 ml, of saline. Next,the cells were dissociated by incubation at room temperature or at 37°C. for 3 to 10 minutes with 1.5 mL of trypsin/EDTA. The action of thetrypsin was stopped by addition of 8 mL of D-MEM/F-12 containing 10%(v/v) FBS or 8 mL of the horse serum or FBS. The cells were collected bycentrifugation at 1000×g for 5 minutes and suspended in 10 mL of freshserum containing medium. Aliquots were diluted into Isoton II (CoulterDiagnostics) and cell numbers determined with a Model ZBI or Z1 CoulterParticle Counter. The new dishes (100-mm diameter with 15 to 20 mL offresh medium) were seeded with 2.0×10⁵ to 1.0×10⁶ cells on analternating three-four day schedule or weekly as dictated by cell linegrowth rate. Cultures were used for growth assays between three and sixdays after passage. Acidic (yellow medium indicator color) cultures arenot used for growth assays.

General Cell Culture—Media Types Used.

The assays done in the presence of serum were initially in “low-Fe”D-MEM/F-12 containing phenol red (Moreno-Cuevas J E and Sirbasku D A(2000) In Vitro Cell Dev Biol 36, 410-427). The issue of thesignificance of the presence or absence of phenol red, a potentialestrogen (Berthois Y et al. (1986) Proc Natl Acad Sci USA 83,2496-2500), has been dealt with in considerable detail (Moreno-Cuevas JE and Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 447-464). The Fe(III) content of this medium was ≦0.2 μM (Eby J E et al. (1992) AnalBiochem 203, 317-325). Fe (III) levels of ≧1.0 μM interfere with thyroidhormone and estrogen responsive rat pituitary tumor cell growth inculture (Eby J E et al. (1992) Anal Biochem 203, 317-325; Eby J E et al.(1993) J Cell Physiol 156, 588-600; Sato H et al. (1991) In Vitro CellDev Biol 27A, 599-602; Sato H et al (1992) Mol Cell Endocrinol 83,239-251). Although Fe (III) might prevent estrogen responsiveness frombeing identified in culture with MTW9/PL2 cells, as shown herein andreported (Sirbasku D A and Moreno-Cuevas J E (2000) In Vitro Cell DevBiol 36, 428-446; Moreno-Cuevas J E and Sirbasku D A (2000) In VitroCell Dev Biol 36, 447-464), this is not the case when serum is present.Standard Fe (III)/Fe (II) containing D-MEM/F-12 was as effective as thelow-Fe medium. It is clear that the apotransferrin in the serumeffectively reduced the free Fe (III) in the medium to less thancytotoxic levels. As stated above, apotransferrin binds Fe (III) withvery high affinity at pH 7.4 in plasma. The total concentration oftransferrin in serum is about 3 mg/mL. Usually, two-thirds of the totalis apotransferrin. This amount is more than adequate to chelate Fe (III)in culture medium (Eby J E et al. (1992) Anal Biochem 203, 317-325).However, in assays in serum-free defined medium, as described below, aFe (III) chelator (e.g. apotransferrin or DFX) is present in theserum-free defined medium at sufficient levels to neutralize the toxiciron.

General Cell Culture—Growth Assay Methods.

Cell growth assays were initiated with stock cultures that wereharvested by trypsin/EDTA treatment as described above with oneexception. It was highly preferred to stop the action of trypsin with 3mL, of soybean trypsin inhibitor (0.5% w/v in saline) instead of mediumcontaining serum. The use of trypsin inhibitor reduced the possibilityof contamination of the subsequent assay media by serum-derived steroidhormones. The dissociated cells were collected by centrifugation asdescribed above and washed three times with 10 mL volumes of serum-freestandard D-MEM/F-12. After each wash, care was taken to aspirate allmedium from the cell pellet and the walls of the centrifuge tubes. Thisminimized the carryover of steroid hormones into the experimental testdishes. By taking steps to avoid carryover of serum, steroid hormonesare prevented from being retained by the cells in culture. It is highlypreferred to wash the cells in this way before assaying to measurevarious steroid hormone effects in culture. It has been reported thatsteroid hormones are retained long term by breast cancer cells inculture (Strobl J S and Lippman M E (1979) Cancer Res 39, 3319-3327).The above-described wash procedure negates this problem. After the finalwash, the cells were suspended in 10 mL of serum-free D-MEM/F-12 andcell numbers determined. When cells were to be assayed in medium withoutphenol red discussed elsewhere herein and reported (Moreno-Cuevas J Eand Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 447-464), the cellswere washed and resuspended in phenol red free D-MEM/F-12 purchased fromGibco-BRL. The growth assays were initiated in 35-mm dishes containing atotal of 2.0 mL of medium and the final concentration of all componentsexcept steroid hormones. The steroid hormone stocks were diluted toappropriate concentrations in serum-free D-MEM/F-12 and 20 μL aliquotsadded to each dish. For all growth assays, the medium was not changedafter the initial inoculation. Because several of the cell linesdescribed in Table 2 grow in serum containing medium and serum-freedefined medium as mixtures of suspension and attached cells, removal orchanging of the medium during the course of the assays causessubstantial cell losses. For all cell growth assays, the initial seeddensities ranged from 5,000 to 12,000 cells per 35-mm diameter dish.

General Cell Culture—Steroid Hormone Preparations.

A number of hormone preparations are used to supplement the cellcultures. Unlabeled steroid hormones were obtained from Sigma orSteraloids. Stock solutions were prepared in sterile glass containers.The powder (non-sterile) steroid is added to the bottle along with 200ml of 70% aqueous ethanol (ready as sterile). The steroids dissolvewithin an hour at room temperature, or when required were dissolved bygentle heating on a hot plate (hand temperature test-no boiling-no openflames). The stock solutions were stored at 4° C. and renewed atsix-month intervals. It is not necessary or desirable to filtersterilize these solutions because of steroid hormone loss on filtermembranes. Stocks of 1.0 mM steroid hormones were prepared. To preparediluted stocks for direct use in culture, 10 μL of 1.0 mM steroidhormone is diluted into 10 mL of D-MEM/F-12. This gives a stock of 1.0μM. It is used in the assay dishes or diluted further in D-MEM/F-12 asneeded. The diluted steroids are discarded after each use because theybind to the plastic with storage. The formula weight (FIAT) of each ofthe common natural and synthetic hormones used is listed below in Table2 along the abbreviation used for each and the amounts required toprepare 200 mL of stock.

TABLE 2 Preparation of Steroid Hormone Stocks for Cell Culture andHormone Binding Assays FORMULA STEROID HORMONES WEIGHT (FW)MILLIGRAMS/200 mL 17β-estradiol (E₂) 272.4 54.4 Estrone (E₁) 270.4 54.1Estriol (E₃) 288.4 57.7 Diethylstilbestrol (DES) 268.4 53.7 TamoxifenCitrate (TAM) 563.6 112.7 Progesterone (PROG) 314.5 62.9Hydrocortisone/Cortisol (C) 362.5 72.5 Dexamethasone (DEX) 392.5 78.5Testosterone (T) 288.4 57.7 Dihydrotestosterone (DHT) 290.4 58.1

General Cell Culture—Harvest and Counting Cells.

At the termination of the experiments, each plate received 0.4 mL ofcrude pancreatic trypsin dissolved in phosphate buffered saline wasadded along with 0.3 mL of 0.29 M EDTA. After 4 to 40 minutes incubationat room temperature or at 37° C., the action of the trypsin was stoppedby addition of 0.6 mL of horse serum. The cell clumps were dissociatedfurther by one passage through a 20½ or 23-gauge needle and syringe.This suspension was then diluted to 10 mL with Isoton II and cellnumbers determined with a Coulter Counter. The results are presented asthe average of triplicate dishes for each test medium. To determine dayzero cell numbers, at least triplicate 1.0 mL aliquots of the inoculumwere collected for counting during the seeding of the test dishes.Coulter Counter standardization and monitoring were performed by themanufacturer.

General Cell Culture—Quantification of Growth.

The cell number results are converted to cell population doublings (CPD)by the following calculation:

${CPD} = \frac{\frac{{Log}_{10}\mspace{14mu}{Average}\mspace{14mu}{Cell}\mspace{14mu}{Number}\mspace{14mu}{on}\mspace{14mu}{Collection}\mspace{14mu}{Day}}{{Log}_{10}\mspace{14mu}{Average}\mspace{14mu}{Cell}\mspace{14mu}{Number}\mspace{14mu}{on}\mspace{14mu}{Day}\mspace{14mu}{Zero}}}{{Log}_{10}2}$

For the purposes of this Disclosure, the mitogenic response to sexsteroid hormones is designated the “steroidogenic effect.” For example,the “estrogenic effect” is calculated as the difference between CPDmeasured in the presence of an estrogen minus CPD in the absence of thesteroid. These values equal cell number increases of 2^(CPD). The term“androgenic effect” has the same meaning except that it describes growthcaused by androgens such as DHT and T. CPD is used herein as a measureof growth because it is a direct calculation of the number of times acell population undergoes cell division. Furthermore, CPD use permits adirect measure of ED₅₀ and ED₁₀₀ Concentrations in different and inreplicate assays. The significance of differences between test dishesand controls was evaluated by the student's t test. Values of p<0.05were accepted as significant. Standard deviations (±SD) are includedwhen appropriate.

Discussion of Example 1

The cell culture methods outlined above are highly preferred asprocedures to obtain cells sufficiently washed of steroid hormone tomeasure low concentration effects in medium with hormone depleted serumprepared as described in the next Example. The use of STI to stop theaction of the trypsin is highly preferred. Application of serum to stopthe action of the trypsin causes a substantial loss of hormoneresponsiveness.

Example 2 Methods of Preparing Steroid Hormone Depleted Serum

In this example, two methods for preparing steroid hormone depletedserum are described. The primary purpose was to prepare serum thatsupported large magnitude sex steroid growth effects in culture and toidentify the dose-response concentrations that cause the effects, asdemonstrated in Examples that follow. This meant preparing serum with ≦5μg/mL estrogen (and other steroid hormones). This concentrationcorresponds approximately to the lower limit of detection of steroids byradio immunoassay. The methods tested included (A) a two-stepcharcoal/dextran extraction of serum at 34° C., and (B) a one steptreatment with Amberlite™ XAD™-4 resin at 37° C.

A. Charcoal-Dextran Extraction at 34° C.

Preparation of the Charcoal/Dextran Mixture.

Activated charcoal, untreated powder (100 to 400 mesh), was obtainedfrom Sigma (Catalog No. C5260). This preparation was done at roomtemperature. The powder (30 g) was suspended in 600 mL of water andstirred for 20-30 minutes at room temperature. The water used to washand suspend the charcoal was a purified source made by reverseosmosis/ion exchange treatment/charcoal filtration/0.2 μm pore diameterfiltration/distillation into glass (only) containers. Next, 3.0 g ofDextran T70 (Pharmacia) was dissolved in 300 mL of water, added to thecharcoal suspension with stirring, and the mixture stirred for 30-60minutes at room temperature, preferably 60 minutes. The mixture was thenwashed with about 6-8 liters of distilled water in a sintered glassfunnel (2000 mL, ASTM 40-60, C#36060). This wash removes impurities aswell as fine particles of charcoal that cannot be separated from serumby centrifugation. The charcoal-dextran retentate was suspended in afinal volume of 300 mL of distilled water to yield a suspension of 100mg/mL charcoal and 10 mg/mL dextran. Preferably the mixture is stirredvigorously for about an hour, and then stored at room temperature for nomore than about 2-3 weeks prior to use.

Charcoal-Dextran Extraction at 34° C. Of Horse Serum (CDE-Horse Serum).

This serum in 500 mL sterile bottles was removed from the freezer (−17°C.) and thawed at 4° C. for 24 to 48 hours. Alternatively, fresh serumcould be used. The thawed serum (still in the 500 mL sterile bottles)was placed in an orbital shaker water bath (Lab-Line Orbit Shaker WaterBath) equilibrated at 34° C. The serum was incubated at 140 RPM for45-60 minutes to reach 34° C. Approximately 250 mL portions of the 34°C. serum (total volume about 1 liter) were transferred to one-literErlenmeyer flasks and tightly capped with aluminum foil. These wereincubated for 20-30 minutes (preferably 30 minutes) in the 34° C.orbital shaker water bath at a medium-high rotation speed. Thereafter,25 mL of the charcoal/dextran suspension was added to each flask. Thecharcoal-dextran suspension was stirred at room temperature whileremoving the 25 mL aliquot. The final charcoal concentration in eachflask was 10 mg/mL, and the final concentration of dextran was 1 mg/mL.After addition of the charcoal-dextran mixture to each flask, theextraction mixtures were shaken at 140-160 RPM at 34° C. for two hours.After this, the mixture was cooled on ice and the charcoal removed bycentrifugation at 10,000×g for about 60 minutes at room temperature. Insome preparations the temperature of the mixture gradually warmed toabout 40° C. during centrifugation. The supernatants were pooled in atwo-liter beaker and 275 mL portions of the supernatant (serum)transferred to fresh one-liter Erlenmeyer flasks. These were thenincubated in the orbital shaker water bath at 34° C. for 20-30 minutes(preferably 30 minutes) to re-equilibrate the temperature. A secondextraction was done by addition of a fresh aliquot (about 14 mL) of thecharcoal-dextran suspension. This re-extraction mixture was incubatedwith shaking for another 2 hours at 34° C. The final charcoalconcentration during this extraction was about 5 mg/mL. Afterward thebulk of the charcoal was removed by centrifugation, as before. In somepreparations the temperature of the mixture reached about 41° C.,without harming the quality of the serum. The supernatants werecollected into a two-liter beaker and filtered through 5 μm porediameter filters to remove residual charcoal. If it was considerednecessary for particular preparations that still contained residualcharcoal, (for example, due to charcoal darkening serum) the serum wasalso filtered with 0.45 μm pore diameterMillipore filters. Thesefiltrations were done with plastic reusable filter holders and lightvacuum. The steroid hormone depleted serum was then sterilized using 0.2μm pore diameter filters. After sterilization, aliquots of about 26 mLwere dispensed into sterile glass (50 mL) bottles or sterile 50 mLpolypropylene tubes and stored frozen at −17° C. Although 34° C. ispreferred in the above-described regime, and provides the best results,satisfactory depletion of steroid hormones can be obtained over thetemperature range of about 30 to 37° C. The 2 hour incubation times forthe extraction and re-extraction mixture (at 34° C.) are preferred, buta time range of 30 minute to 3 hours could also be used with success,employing longer incubation times for the lower temperatures within the30-37° C. range. A±25% variation in the charcoal concentration used foreach extraction had no detrimental effects on the final product.

B. Amberlite™ XAD™-4 Resin Treatment.

In a different procedure carried out to free corticosteroids bindingglobulin (CBG) of storage cortisol, XAD resin has been used to removethe steroid by incubation for 5 hrs at room temperature (A. M. Nakhla,et al. (1988) Biochem. Biophys. Res. Commun. 153, 1012-1018). Describedas such, this method removed only about 80% of cortisol from thepurified protein. Careful application of that method failed to yieldserum suitable for the purpose of this study. As an alternative topreparing steroid hormone depleted serum by charcoal-dextran extraction,horse serum was treated by incubation with Amberlite™ XAD™-4 nonionicpolymeric absorbent (Aldrich, Catalog. No. 21, 648-8; or Sigma. CatalogNo. XAD-4 37380-42-0). Specifically, a 500 mL bottle of horse serum wasthawed at 37° C. and divided into 250 mL portions that were each in aone-liter Erlenmeyer flask. To each flask was added 25 grams of moistXAD-4™ resin. The mixtures of serum and resin were then incubated withshaking in a rotary Labline Orbital Shaker water bath at 34° C. at abouttwo-thirds of the maximum rate for 24 hours (speed adjusted to controlfoaming). This extraction can be done at temperatures from 30° C. to 37°C. At 30° C., the extraction requires 24 to 36 hours. At 37° C., itrequires 18-24 hours to be complete. The 34° C. and 37° C. proceduresare preferred. Each flask was tightly capped with aluminum foil andtaped. After 24 hrs, the resin is allowed to settle by gravity, thesupernatant decanted, and then vacuum filtered using a glass fiberfilter in a Buchner funnel. The resulting serum was filter sterilizedusing 0.2 μm pore filter units. Aliquots of about 26 mL were frozen at−17° C. in 50 mL sterile bottles or 50 mL polypropylene tubes.

Discussion of Example 2

Each of the methods presented have advantages, depending on theparticular needs and desires of the user. The scale procedures describedare useful to prepared sufficient serum for testing of plasma or bodilyfluids samples for inhibitors and for hormone activities or anti-hormoneactivities or evaluation of toxicity of compounds in cell cultureassays. To ensure uniformity, large batches of the serum can beprepared, if desired. The charcoal method described above is readilyapplicable to one to five liter volumes of serum per preparation. Withuse of moderate numbers of test samples or ≦50 mL per test substance,this is an adequate supply. To prepare larger volumes of serum (i.e. ≧20liters) for extensive testing programs or commercial applications, thecharcoal-dextran methods will preferably employ industrial filtration orother separation equipment to remove the carbon after each extraction.The XAD-4™ resin method as presented is adaptable to one to five litersfor testing purposes. For industrial applications, where ≧20 to 100liter batches are customarily required, the resin method is preferredbecause of the need for only one separation after extraction. However,where “foaming” of the serum protein is to be avoided completely,charcoal extraction is superior. The materials cost for charcoal-dextranhas an advantage when economy is a major consideration. It is lessexpensive than XAD-4™ resin on a per liter basis, although the resin iscommercially available at low cost when purchased in large amounts (i.e.≧50-100 kilograms). XAD-4™ resin method is highly adaptable to smallclinically derived samples of plasma or other bodily fluids.

This 34° C. method has been used to prepare CDE human serum, porcineserum, rat serum, hamster serum, ovine serum, fetal bovine serum, newborn bovine serum (0 to 10 days old), young donor bovine serum (10 daysto 6 moths old) young adult bovine serum (300 to 900 lbs), fetal horseserum, chicken serum, turkey serum, dog serum, goat serum, rabbit serumand monkey serum. Subsequent Examples demonstrate how these strippedsera are preferably employed. The results demonstrate the broad utilityof the method of preparing charcoal-dextran extracted serum for testingof cell lines from many species using homologous serum assays. Fromthese results it can also be readily appreciated that these methods areapplicable not only to testing of human plasma/serum, but also toveterinary medicine samples or compounds of significance to domesticanimals, as well as any application where a steroid hormone strippedserum is used. For example, in the diagnosis, prevention/risk managementor therapy of mucosal origin cancers.

Example 3 MTW9/PL2 Rat Mammary Tumor Cells

This Example describes a sensitive in vitro model assay system fordetecting and measuring steroid hormone responsive cell growth.

The MTW9/PL2 Rat Mammary Tumor Cell Line.

The properties of the MTW9/PL2 have been summarized (Moreno-Cuevas J Eand Sirbasku D A In Vitro Cell Dev Biol 36, 410-427). The MTW9/PL cellline was established by our laboratory in culture from thecarcinogen-induced hormone-responsive MT-W9A rat mammary tumor of a W/Furat. This tumor formed estrogen, androgen and progesterone responsivetumors in W/Fu rats (Sirbasku D A (1978) Cancer Res 38, 1154-1165). Itwas later used to derive the MTW9/PL2 cell population that was alsoestrogen-responsive in vivo (Danielpour D et al. (1988) In Vitro CellDev Biol 24, 42-52). In serum supplemented culture conditions theMTW9/PL2 cells demonstrate 80-fold steroid hormone growth responses. Allsera used were steroid hormone-depleted by charcoal-dextran treatment at34° C. The studies were done with horse serum as well as serum fromother mammalian species. The growth of the MTW9/PL2 cells was biphasicin response to hormone-depleted serum. Concentrations of ≦5% (v/v)promoted optimum growth. Above this concentration, serum was inhibitory.Concentrations ≧40% (v/v) inhibited growth altogether. Addition of1.0×10⁻¹³ to 1.0×10⁻⁸ M 17-estradiol (E₂) reversed the inhibitioncompletely. At 1.0×10⁻⁸ M, E₁, E₃ and DES promoted growth as well as E₂.Testosterone and DHT promoted growth only at 10⁻⁷ M. Progesterone waseffective at 10⁻⁶M. Cortisol was ineffective. Labeled hormone bindinganalysis and Western immunoblotting documented that MTW9/PL2 cells hadestrogen and progesterone receptors but not androgen or cortisolreceptors. Estrogen treatment of MTW9/PL2 cells induced a concentrationand time dependent increase in progesterone receptors. It was concludedthat (1) the MTW9/PL2 population is the first highly steroid hormoneresponsive rat mammary tumor cell line to be established in culture froma carcinogen induced tumor and (2) sera from a number of speciesincluding horse, rat and human contain an inhibitor which mediatesestrogen sensitive MTW9/PL2 cell growth in culture.

Estrogenic Effects with MTW9/PL2 Rat Mammary Tumor Cells in CulturesSupplemented with CDE-Horse Serum.

Unless otherwise stated, references in this and the following Examplesto “CDE-horse serum” refer to the 34° C. charcoal-dextran extractionprocess described in above. The MTW9/PL2 cells were assayed for E₂responsiveness in cultures supplemented with increasing concentrationsof CDE-horse serum (FIG. 1A). Concentrations ≦5% (v/v) promoted growth.Typically within seven days cell numbers increased from 6,000 per dishto more than 200,000 in 2 to 5% serum. This most likely resulted fromstimulation by serum-borne growth factors as well as the mitogeniceffect of transferrin (Danielpour D et al (1988) In Vitro Cell Dev Biol24, 42-52; Riss T L and Sirbasku D A (1987) In Vitro Cell Dev Biol 23,841-849; Riss T L et al. (1986) J Tissue Culture Methods 10, 133-150).As serum concentrations exceeded 5% (v/v), the effects of the growthpromoters were counteracted by a serum-borne inhibitor(s). At serumconcentrations of 30 to 50% (v/v), growth was completely inhibited.Usually only seed density cell numbers were found after seven days incultures containing 50% (v/v) CDE-horse serum. In contrast, the presenceof 1.0×10⁻⁸ M E₂ completely reversed the serum dependent inhibition. Incultures supplemented with 20 to 50% (v/v) CDE-serum plus 1.0×10⁻⁸ M E₂,cell numbers were 400,000 per dish. Logarithmic quantifying of cellgrowth was done by converting the cell number data in FIG. 1A into CPD.A plot of these values is shown in FIG. 1B. The estrogenic effect isalso presented. In FIG. 1B, the difference was maximum at 30% (v/v)CDE-horse serum. It was a 6.14 CPD or a 70-fold (i.e. 2^(CPD) or2^(6.14)) increase in cell numbers in response to E₂. In randomlyselected replicate experiments (N=9) done over a two-year period withdifferent preparations of CDE-horse serum, the average maximum estrogeneffect±SEM was 6.43 CPD±0.49 (range 5.63 to 7.22). This was an 86-fold(2^(6.43)) estrogenic effect. The modal concentration of serum thatpromoted maximum E₂ effects was 40% (range 20 to 50%).

Estrogen Reversibility of the Growth Inhibition Caused by CDE-HorseSerum.

It was examined whether inhibition caused by CDE-horse serum wasreversible even after several days in culture (FIG. 2). The MTW9/PL2cells were seeded into medium containing 50% (v/v) CDE-horse without E₂and cell numbers monitored daily. Growth ceased within 48 hours;thereafter cell numbers remained static. In parallel cultures, additionof E₂ on days two, four, and six after seeding caused resumption ofgrowth (after a lag period) at nearly the same rate as cultures thatreceived hormone at the time of inoculation. These results show that thecells survived in the presence of the inhibitor without E₂ for at leastsix days. As described in a later Example, longer exposure to thepurified inhibitors was cytotoxic and suggested therapeutic value.

Storage Stability of CDE-Horse Serum.

In Table 3, the effect of storage temperature on the estrogen mediatingactivity of CDE-horse serum is shown. The assays were done with MTW9/PL2cells as shown in FIGS. 1A and 1B. Stability was assessed by fourcriteria: (i) the concentration of serum needed to give an estrogeniceffect of 2.5 CPD (i.e. ED_(2.5)), (ii) the percent serum needed for themaximum estrogenic effect, and the magnitude of the estrogenic effects(CPD) at (iii) 20% and (iv) 30% serum. CDE-horse serum was stable at 23°C. for three weeks without loss of activity as assessed by all fourcriteria. Storage at 4° C. was detrimental within 24 days as measured bythe CPD at 20% and 30% (v/v) serum concentrations. Longer storage at 4°C. was not advisable. Storage at −17° C. was most effective; theactivity was unchanged even after 90 days. In experiments not shown,repeated freeze-thaw cycles caused an appreciable loss of inhibitoractivity. The results in Table 3 show that serum stored frozen hasutility for long periods and therefore provides a stable supply fortesting of clinically relevant samples. Also, it is clear that clinicalsamples to be assayed for inhibitor can be stored for a few days at roomtemperature without damage.

TABLE 3 Summary of the Effects of Serum Storage Temperature on Activity.% Serum needed Maximum E₂ CPD at Days for 2.5 CPD Induced CPDs 20% of(ED_(2.5)) of E₂ (% serum, v/v, for (v/v) CPD at 30% Storage Inducedgrowth the maximum) serum (v/v) serum Storage at 23° C. 1 2.1 4.9 (10%)5.0 3.2 3 5.2 5.4 (20%) 6.2 5.2 6 5.0 4.2 (10%) 3.5 0.9 14 2.9 6.0 (10%)4.3 2.6 23 4.0 6.3 (10%) 3.9 2.5 Storage at 4° C. 1 1.8 5.9 (10%) 4.94.0 7 6.8 5.7 (20%) 6.4 5.4 15 3.8 4.1 (30%) 5.5 4.2 24 5.3 5.3 (10%)1.0 2.8 44 3.0 4.8 (5%) 0.04 0.26 55 2.2 5.0 (5%) 0.00 0.24 90 >50 2.1(5%) 0.30 0.40 Storage at −17° C. 1 2.6 5.2 (10%) 5.0 3.1 7 4.0 5.8(30%) 6.8 5.8 44 3.3 5.8 (20%) 6.0 5.4 90 6.1 5.2 (30%) 6.2 5.9

Dose-Response Effects of Steroid Hormones in CDE-Horse Serum.

The dose effects of a number of steroid hormones were evaluated withMTW9/PL2 cells in medium containing 50% (v/v) CDE-horse serum. Theresults of one of these studies (N=3) are presented in FIG. 3. Estrogenswere the most effective mitogens. Their order of potency was E₂>E₁>E₃.This relative potency was expected based on the affinities of thesesteroids for the estrogen receptors of other target tissues (Clark J Hand Markaverich B M (1983) Pharmacol Ther 21, 429-453). The cell numbersin dishes containing 1.0×10⁻¹³ M E₂ were 32-fold (p<0.01) higher than indishes without the hormone. Concentrations of 1.0×10⁻¹² to 1.0×10⁻¹¹ ME₂ promoted 6.73 CPD that was a 110-fold estrogenic effect in sevendays. The ED₅₀ of E₂ was about 0.5 to 1.0×10⁻¹² M. Using E₁ and E₃,optimum growth was achieved at 1.0×10⁻⁹ and 1.0×10⁻⁸ M, respectively. Inexperiments not shown, the mitogenic potency of the synthetic estrogenDES was assessed. At 1.0×10⁻⁸ M, it caused the same growth as saturatingconcentrations of the natural estrogens. The DES effect was 6.98 CPD inseven days that was a 126-fold (2^(6.98)) increase in cell number. Thenext most potent hormone was DHT. It caused significant (p<0.05) growthat supraphysiologic concentrations ≧1.0×10⁻⁸ M. Progesterone also causedsignificant growth, but only at supraphysiological concentrations≧1.0×10⁻⁷ M. Cortisol was ineffective at concentrations up to 1.0×10⁻⁶M.

Estrogen Mitogenic Effects with MTW9/PL2 Cells in CDE-Serum from SeveralSpecies.

Serum from species other than horse were examined to determine they alsopossessed estrogen reversible inhibitory activity with rat MTW9/PL2cells. These experiments are shown in FIG. 4. All of the sera testedwere charcoal dextran extracted at 34° C. CDE-porcine (FIG. 4A), andCDE-human serum (FIG. 4B) showed patterns nearly identical to that ofCDE-horse serum. The maximum estrogenic effects with both sera were sixto seven CPD (N=3). CDE-rat serum also showed the same pattern ofestrogen reversible growth inhibition (FIG. 4C). CDE-ovine serum showedestrogen reversible inhibition equivalent to CDE rat serum (data notshown). With serum from rats, the maximum estrogenic effect was four tofive CPD (N=4). CDE-bovine serum (adult donor herd) displayed the samepattern of activity as other sera (FIG. 4D). CDE-fetal bovine serumshowed a different pattern (FIG. 4E). Even at 40% (v/v), there was noinhibition. With some batches of this serum, there was no inhibitioneven at 50% (N=2). With others (N=2), inhibition was found. In theseexperiments, the estrogenic effects reached three to four CPD in 50%(v/v) CDE-serum. Even with this variability, fetal bovine serum has lessactivity than the serum from the adults of this species. The assays withCDE-fetal horse serum (N=3) showed inhibition at 50% (v/v) that was notreversible by 10 nM E₂ (FIG. 4F).

Discussion of Example 3

The MTW9/PL2 Cell Line as a Unique Rodent Test System.

The present study shows very clearly that (ER⁺) MTW9/PL2 cells areestrogen growth sensitive in culture and applicable to testing of serumor bodily fluid inhibitors or sex steroids in such preparations. Theestrogen receptor content and estrogen affinity characteristics of theMTW9/PL2 cells indicate appropriate stability for commercialapplications. The MTW9/PL2 population is the first highly steroidhormone-responsive rat mammary tumor cell line to be established inculture from a carcinogen-induced tumor”. As a direct consequence of theinformation provided above, this cell line is a unique and valuableasset for combination in vitro and in vivo modalities to be applied toclinically and commercially significant compounds or preparations andfor the assay of the inhibitor content or hormone or anti-hormoneactivities.

Technical Conditions for Demonstrating Estrogen Responsiveness inCulture and Evidence for a Serum-Borne Inhibitor.

Conditions that permit the observation of very large magnitude estrogenmitogenic effects with the permanent MTW9/PL2 cell line in culture aredefined herein. As mentioned in the Background of the Invention, mostexisting rat mammary tumor cell lines are not suitable for use inevaluating hormone responsiveness in vivo because they are derived fromoutbred animals. This problem was overcome by developing the MTW9/PL2rat mammary tumor cell line from a carcinogen-induced hormone responsivetumor (i.e. the MT-W9A tumor), first induced and transplanted in aninbred W/Fu rat as described (MacLeod R M et al. (1964) Cancer Res 75,249-258). The MTW9/PL2 cells form hormone responsive tumors whenimplanted in these rats (Sirbasku D A (1978) Cancer Res 38, 1154-1165;Danielpour D and Sirbasku D A (1984) In Vitro 20, 975-980; Riss T L etal. (1986) J Tissue Culture Methods 10, 133-150). In culture, theMTW9/PL2 cells showed the same hormone responsiveness expected of ratand human breast epithelial cells, as shown herein and subsequentlyreported (Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell DevBiol 36, 410-427; Sirbasku D A and Moreno-Cuevas J E (2000) In VitroCell Dev Biol 36, 428-446; Moreno-Cuevas J E and Sirbasku D A (2000) InVitro Cell Dev Biol 36, 447-464).

The effects of the steroid hormones in culture were the same asdescribed for the growth of the original MT-W9A tumor in W/Fu rats(MacLeod et al. (1964) Endocrinology 75, 249-258) and tumor formation bythe parental MTW9/PL cell line in this same strain of rats (Sirbasku D A(1978) Cancer Res 38, 1154-1165). The present embodiment is the firstestablished cell line derived from a carcinogen induced rat mammarytumor that continues to show large magnitude growth responses toestrogens, progesterone and androgens even after extended periods inculture, preferably when cultured under the conditions disclosed herein.Thyroid hormone responsiveness has also been demonstrated for MTW9/PLcells (Leland F E et al. (1981) In: Functionally Differentiated CellLines, Sato G, ed, Alan Liss, New York, pp 1-46). Of the other importanthormones known to influence the growth of the original MT-W9A tumor,only prolactin remains to be investigated. Prolactin is not mitogenicfor the parental MTW9/PL cells under serum-free defined conditions(Danielpour D et al. (1988) In Vitro Cell Dev Biol 24, 42-52).Continuing investigations are directed toward evaluating the possibilitythat prolactin also reverses the effects of the serum-borne inhibitor orotherwise acts as a cytokine to influence the production ofimmunoglobulins in breast and other mucosal tissues. The development ofthis cell line now permits not only sensitive steroid hormone growthanalysis in culture, but also direct comparisons to the effectiveness ofthe same test substances in animals. No other such rat mammary system iscurrently available.

MTW9/PL2 Receptor not Lost in Culture.

The present results showing an average 86-fold MTW9/PL2 cell numberincrease in seven days in response to physiological concentrations of E₂have several important technical implications. Most notably, theycontradict many earlier explanations for why estrogen stimulated cellgrowth has been difficult to demonstrate in culture. Originally, thelack of estrogenic effects in culture was thought to be due to adedifferentiation of cells that resulted in a loss of functionalreceptors or some other aberration that disrupted the growth response.In light of the present Disclosure, this explanation now seems veryunlikely. The present results show the presence of similar levels ofestrogen receptors in both the original MTW9/PL cell line reported in1982 and the current MTW9/PL2 cells. Analyses made by others showingestrogen receptors in established cell lines in culture (Horwitz K B etal. (1978) Cancer Res 38, 2434-2437; Haug E (1976) Endocrinology 104,429-437; Soto A M et al. (1988) Cancer Res 48, 3676-3680; Keydar I etal. (1979) Eur J Cancer 15, 659-670; Engel L W et al. (1978) Cancer Res38, 3352-3364) also mitigate against this explanation. Furthermore, theestrogen receptors of the MCF-7 cells were functional based on thedemonstration of estrogen inducibility of the progesterone receptor(Horwitz K B and McGuire W L (1978) J Biol Chem 253, 2223-2228). As withthe human breast cancer cells, the MTW9/PL2 line was also significantlyestrogen responsive by this criterion. When all of the available data isconsidered in light of the presently disclosed observations, the notionthat long-term culture necessarily leads to loss of functional estrogenreceptors is laid to rest. A major advantage of the MTW9/PL2 line is itslong-term stability permitting series analyses over long periods of timewithout concern for cell property changes.

Prolonged Steroid Hormone Retention by Culture Cells.

It has been suggested that prolonged retention of estrogens might be thereason for a lack of responsiveness of target cells in culture (Strobl JS and Lippman M E (1979) Cancer Res 39, 3319-3327). Investigators havereported that the half-life of loss of specifically bound ³H-E₂ fromMCF-7 cells was about 24 hours at 37° C. (Strobl J S and Lippman M E(1979) Cancer Res 39, 3319-3327). Cells from stock cultures grown inuntreated/steroid hormone containing serum were proposed to retainstimulating levels of estrogens. Even several washes over 78 hours didnot attenuate the problem (Strobl J S and Lippman M E (1979) Cancer Res39, 3319-3327). Conversely, the studies herein did not identify thisproblem. All the assays reported here were done with cells takendirectly from cultures grown in steroid hormone containing serum (e.g.fetal bovine serum). After trypsinization of the MTW9/PL2 cells fromstock culture, only three careful washes with serum-free D-MEM/F-12 wereperformed before initiating the growth assays. The results in FIG. 3show clearly that 1.0×10⁻¹² M E₂ caused significant MTW9/PL2 cellgrowth. Also, the results in FIG. 2 show that MTW9/PL2 cells ceaseproliferation within 48 hours of starting a growth assay. Theseobservations either support the conclusion that prolonged steroidhormone retention by cells is not as serious an issue as first proposedor are evidence that the technical processes described herein to preparecells for assays have eliminated this problem. With regard to thepresent investigation, all cell lines studied showed this same propertywhen prepared by the same technical process for growth assays.

Merits of Charcoal Extraction.

Other investigators have challenged the use of charcoal extraction todeplete serum of steroid hormones. It has been stated that thisprocedure absorbs or otherwise alters serum to make it ineffective(Amara J F and Dannies P S (1983) Endocrinology 112, 1141-1143; Wiese TE et al. (1992) In Vitro Cell Dev Biol 28A, 595-602). To counter thisproblem, either individual lots of untreated serum were used to seekestrogenic effects (Wiese T E et al. (1992) In Vitro Cell Dev Biol 28A,595-602), or serum was prepared from animals after endocrine ablationsurgery (Amara I F and Dannies P S (1983) Endocrinology 112, 1141-1143).One of the best examples of use of surgically depleted serum came fromthe study of the GH₄C₁ rat pituitary cells (Amara J F and Dannies P S(1983) Endocrinology 112, 1141-1143). They were highly E₂ responsive inmedium supplemented with the serum from a gelded horse (Amara J F andDannies P S (1983) Endocrinology 112, 1141-1143). However, experiencewith serum derived by these methods has not been as positive. Forexample, this issue was investigated in 1976 with the related GH₃C₁₄ ratpituitary tumor cell line (Kirkland W L et al. (1976) J Natl Cancer Inst56, 1159-1164), and found that serum from ovariectomized sheep oradrenalectomized and ovariectomized sheep did not support estrogeneffects. Furthermore, unextracted sera from different species can attimes support limited estrogenic effects. However, the estrogeniceffects are of lower magnitude than those in the CDE-serum describedherein. The results are so variable that they typically exclude use as aclinical testing assay. Based on the observation that CDE-serum from anumber of species was very effective, it seems highly unlikely that thenow-disclosed preferred 34° C. procedure is deleterious. However, it isclear from other studies that the 56° C. charcoal method caused atemperature dependent loss of the inhibitor (data not shown). Thepresently described CDE-serum provides greater consistency andreproducibility than the other proposed approaches (Amara J F andDannies P S (1983) Endocrinology 112, 1141-1143; Wiese T E et al. (1992)In Vitro Cell Dev Biol 28A, 595-602). Another advantage is that theseresults do not dependent significantly on the lot of serum purchased.Furthermore, CDE-serum consistently provides larger magnitude estrogeniceffects than serum obtained by either of the other approaches discussedabove.

Steroid Hormone Conjugates are Non-Problematic.

While charcoal treatment can be expected to remove the major classes ofsteroid hormones from serum, there is a question about its effect on themore soluble and potentially active conjugates. It has been reportedthat hydrolysis of estrogen sulfates provided free estrogens in humanbreast cancer cell cultures (Vignon F et al. (1980) Endocrinology 106,1079-1086). This abrogated the effects of exogenous E₂. Although theprevious investigations did not address estradiol sulfate, it was shownthat more than 95% of estrone sulfate and estradiol glucuronide wereremoved from serum by a single 56° C. charcoal extraction (Sirbasku D Aand Kirkland W L (1976) Endocrinology 98, 1260-1272). Additionally, inprevious studies MTW9/PL cells were incubated with tritium labeledestradiol glucuronide for up to 24 hours under cell culture conditionsand found no organic solvent extractable free steroid. Both past andcurrent results indicate that the impact of the estrogen conjugates hasbeen overestimated. In the present study, no precautions were taken toremove the conjugated forms of estrogens from any of the sera tested.Despite this, it was found that many different types of serum wereeffective after charcoal extraction at 34° C. Thus, it is concluded thatremoval of steroid conjugates by digestion or any procedure beyondcharcoal treatment is unnecessary. This is a further advantage of thenew 34° C. method because the additional treatment to remove the steroidconjugates could be prohibitively expensive for larger scale productionthan a few liters, and could potentially introduce undesirable effectsin the serum.

Plastic Product Use for Cell Culture.

The present studies were done with plastic ware made of polystyrene.Plastic is manufactured using alkylphenols (Platt A E (1978) In:Encyclopedia of Chemical Technology, Kirk R E, Othmer D F, eds, 3^(rd)Edition, Volume 26, Wiley, New York, pp 801-847). One of thesecompounds, p-nonyl-phenol, has been reported to be estrogenic for MCF-7cells in culture (Soto A M et al. (1991) Environ Health Perspect 92,167-173). This xenobiotic most likely is present in the cultures used inthese studies. No precautions were taken to exclude compounds leachingfrom plastic. In fact, the bioassay procedures herein are done withpolystyrene plastic ware and culture dishes almost exclusively. If therehad been a significant contamination of the medium by such compounds,the estrogenic effects reported in this study should not have been seenor should have been markedly attenuated. An advantage of the assaysystems described herein is that they have no need for expensive and orexotic substitutes for the common plastic ware used in cell culturelaboratories to conduct bioassays. Also, the CDE-serum can be stored andshipped for commercial use in conventional plastic containers withoutconcern for creation of plastic-induced artifacts. Clinical samples forassay can also be stored and shipped in common plastic ware.

Example 4 Estrogen Responsive Growth of Additional Rodent and Human CellLines in 34° C. Charcoal-Dextran Extracted Horse and Human Serum

In addition to the above-described studies using the MTW9/PL2 ratmammary tumor cell line, several other cell lines were employed todefine the conditions for demonstrating estrogen and androgen responsivecell growth. Established cell lines from a number of different steroidhormone target tissues were selected for growth regulation analysisunder those defined conditions. Additional model cell growth assays formeasuring steroid hormone responsive cell growth are described.

Estrogen Mitogenic Effects with Established ER⁺ Rodent Tumor and HumanCarcinoma Cells in CDE-Horse Serum.

In the first study of this series, the three GH rat pituitary tumor celllines were examined for estrogenic effects in CDE-horse serum. This wasconsidered important in light of their clear responsiveness to manyhormones (Tashjian A H Jr (1979) Methods Enzymol 58, 527-535).Furthermore, these cells are from a tissue that grows coordinately withmammary tissue in castrated rats administered exogenous estrogens. Asdescribed above, this suggested a common regulation mechanism. FIG. 5shows an estrogenic effect ≧5 CPD with GH₄C₁ cells in 10 days. Theresults with GH₃ and GH₁ cells ranged between 4.0 and 5.2 CPD in 10 to14 day assays (data not shown). The same progressive estrogen reversibleCDE-serum inhibition was demonstrable with both rat mammary and ratpituitary tumor cells in CDE-horse serum. To confirm the effectivenessCDE-horse serum with human cells, the ZR-75-1 breast cancer line wasselected because of previous attempts to demonstrate its estrogenresponsiveness in culture (Allegra J C and Lippman M E (1978) Cancer Res38, 3823-3829; Darbre P D et al. (1984) Cancer Res 44, 2790-2793; DarbreP et al. (1983) Cancer Res 43, 349-355). The ZR-75-1 cells showed thesame CDE-serum caused estrogen reversible inhibition as seen with rodentcell lines in this serum. In 14 days, there was a 3.65 CPD 12.5-fold)estrogenic effect (FIG. 6). This was a greater response than recorded inthe ZR-75-1 cell studies cited above. Of all of the cell lines studied,the MCF-7A was the least estrogen responsive even in 50% CDE-horse (FIG.7). The estrogenic effect was 2.65 CPD in 10-12 days. This was stillsignificant (p<0.01) as a 2^(2.65) or 6.3-fold increase in cell numbercaused by estrogen. The present serum-derived inhibitor exhibitedbiological activity exactly opposite the estrogen reversible inhibitorsdescribed by M Tanji et al. (Tanji M et al. (2000) Anticancer Res. 20,2779-2783; Tanji M et al. (2000) Anticancer Res. 20, 2785-2789).

Additional Cell Lines Evaluated.

Evidence is presented herein that the MCF-7K, T47D, LNCaP, and H301cells are highly sex steroid hormone responsive in CDE-horse serum.

Kinetics of Estrogen Responsive Growth in CDE Serum Containing Medium.

In the experiments presented in FIGS. 8A and 8B, ER⁺ cell growth wasmeasured daily for 15 days to determine cell growth kinetics±E₂. Theresults with the T47D line are presented as characteristic of humancells. When evaluated in medium with partially inhibitory 20% (v/v) CDEhorse serum, the effect of E₂ on cell number increase was not apparentuntil after 4 days (FIG. 8A). Increasing the concentration of CDE serumto 50% (v/v) further delayed the effect of E₂ (FIG. 8B). Clearly,whatever mechanism is proposed for the action of the steroid hormone, ittakes a significant period to reverse the effects of the inhibitor. Thisprocess cannot be simply due to a rapid effect on transcription causedby steroid hormones. The interaction of ³H-E₂ with intracellularestrogen receptors saturates in ≦1 hour at 37° C. (Horwitz K B andMcGuire W L (1978) J Biol Chem 253, 8185-8191; MacIndoe H I et al.(1982) Steroids 39, 245-258; Moreno-Cuevas J E and Sirbasku D A (2000)In Vitro Cell Dev Biol 36, 410-427), while de novo hormone inducedprotein synthesis requires only a few hours (Beato M (1989) Cell 56,335-344). Based on a growth lag of ≧4 days, it is likely that steroidhormones initiate a cascade of signaling events that are more complexthan recognized today. To demonstrate that the lag period was related tothe inhibitor, T47D growth was monitored daily in D-MEM/F-12supplemented with 10% (v/v) fetal bovine serum (FIG. 8A). Thisconcentration of fetal bovine serum shows no inhibitor (Moreno-Cuevas JE and Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 410-427). Cellgrowth in medium with fetal bovine serum showed at most a one or two daylag period.

Effect of CDE-Human Serum on Estrogen Responsive Cell Growth.

The next study examined whether human serum was a source of inhibitorfor steroid hormone sensitive cell lines from different species andtissues. The results confirm that CDE-human serum contains approximatelythe same level of inhibitor as CDE-horse serum. Results are shown withT47D human breast cancer cells (FIG. 9A), LNCaP human prostaticcarcinoma cells (FIG. 9B), MTW9/PL2 rat mammary tumor cells (FIG. 9C),two GH rat pituitary tumor cell lines (FIGS. 9D and 9E), and the Syrianhamster H301 kidney tumor cells (FIG. 9F). All lines showed the samebiphasic response to CDE-human serum. Low concentrations (i.e. ≦10%)promoted growth whereas higher concentrations (i.e. ≧20%) progressivelyinhibited growth. Only the absolute magnitudes of the estrogenic effectsvaried. Replicate assays with MCF-7A, MCF-7K and ZR-75-1 cells gave thesame outcomes (data not shown). The experiments reported thus far hereinsupport the conclusion that the inhibitor is ubiquitous in mammals andis not species specific, also subsequently reported (Sirbasku D A andMoreno-Cuevas (2000) In Vitro Cell Dev Biol 36, 428-446).

Dose-Response Effects of Steroid Hormones with Human Breast Cancer Cellsin CDE Serum.

The studies presented thus far have assessed estrogen effects using 10nM E₂. Although 10 nM saturates growth, it is decidedly at the highboundary of physiological. It is important to note that circulatingestrogens in non-pregnant females are generally thought to be in therange of 10⁻⁸ to 10⁻¹⁰ M (Clark J H et al. In: Williams Textbook ofEndocrinology (1992), Saunders, Philadelphia, pp 35-90). Tissueconcentrations are generally conceded to be lower due to SHBG thatreduces the “free” or “active” form of sex steroid hormones (Rosner W(1990) Endocr Rev 11, 80-91). The next studies with T47D cellsdetermined the effective concentration ranges for the three most commonestrogens and compared these to non-estrogen steroid hormones. FIG. 10shows an analysis with T47D cells in D-MEM/F-12 containing 50% (v/v) CDEhorse serum for 14 days. Estrogens were the only physiologicallyrelevant activators of T47D growth. As expected from previous studieswith breast cancer cells (Lippman M E et al. (1977) Cancer Res 37,1901-1907; Jozan S et al. (1979) J Steroid Biochem 10, 341-342;Katenellenbogen B S (1980) Annu Rev Physiol 42, 17-35) and otherestrogen target tissues (Clark J H and Markaverich B M (1983) PharmacolTher 21, 429-453), their order of effectiveness was E₂>E₁>E₃. E₂ causedsignificant (p<0.05) growth when present at 1.0×10⁻¹⁴ M and optimumgrowth at 1.0×10⁻¹⁰ M. Higher concentrations were not inhibitory. TheED₅₀ concentration of E₂ was ≦1.0×10⁻¹³ M. It is noteworthy that even E₃was remarkably potent. Others also had commented that E₃ was more potentthan expected (Lippman M E et al. (1977) Cancer Res 37, 1901-1907). Thisobservation may have special significance because breast cancers thatappear during pregnancy can be particularly life threatening. Humanmaternal plasma has greatly elevated levels of E₃ during the lasttrimester of pregnancy. Testosterone and DHT promoted growth but only atsupraphysiological concentrations (FIG. 10). Other investigators havesuggested that supraphysiological concentrations of androgens actthrough the ER of human breast cancer cells (Zava D T and McGuire W L(1978) Science (Wash DC) 199, 787-788). However, another group hasreported no effect of androgens on human breast cancer cellproliferation (Soto A M and Sonnenschein C (1985) J Steroid Biochem 23,87-94). In the present study, progesterone and cortisol were completelyineffective with T47D cells (FIG. 10). Others have also reportednegative results with these hormones and human breast cancer cells(Schatz R W (1985) J Cell Physiol 124, 386-390; Soto A M andSonnenschein C (1985) J Steroid Biochem 23, 87-94). The data presentedin this Disclosure support the conclusion that the new CDE serum cultureconditions yield physiologically relevant information.

Dose-Response Effects of Steroid Hormones with Rat Pituitary Tumor Cellsin CDE Serum.

The GH family of related cell lines responds to a number of differentclasses of hormones (Amara J F and Dannies P S (1983) Endocrinology 112,1141-1143; Tashjian A H Jr et al. (1970) J Cell Biol 47, 61-70; TashjianA H Jr (1979) Methods Enzymol 58, 527-535; Haug E (1979) Endocrinology104, 429437; Schonbrunn A et al. (1980) J Cell Biol 85, 786-797;Sorrentino J M et al. (1976) J Natl Cancer Inst 56, 1159-1164; RamsdellJ S (1991) Endocrinology 128, 1981-1990; Hayashi I et al. (1978) InVitro 14, 23-30; Faivre-Bauman A et al. (1975) Biochem Biophys ResCommun 67, 50-57). These cells also form steroid hormone responsivetumors in W/Fu rats (Sorrentino J M et al. (1976) J Natl Cancer Inst 56,1149-1154). The GH₄C₁ strain was selected as an example for this nextstudy because of its marked E₂ responsiveness in culture (Amara J F andDannies P S (1983) Endocrinology 112, 1141-1143; Sirbasku D A andMoreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446; Sato H etal. (1991) In Vitro Cell Dev Biol 27A, 599-602) and estrogen requirementfor tumor formation in rats (Riss T L and Sirbasku D A (1989) In VitroCell Dev Biol 25, 136-142). The dose-response effect of steroid hormoneswith GH₄C₁ rat pituitary tumor cells in 50% CDE-horse serum was analyzednext. FIG. 11 shows the results of these experiments. All three majorestrogens promoted growth. The potencies of E₂ and E₁ were equivalentwhereas E₃ was substantially less effective. Even at supraphysiologicconcentrations, E₃ did not promote the saturation densities seen with E₂and E₁. The lowest concentration of E₂ and E₁ that gave significant(p<0.05) growth was 1.0×10⁻¹²M. The ED₅₀ of E₂ was ≦1.0×10⁻¹¹ M. Optimumgrowth required supraphysiological concentrations (i.e. 1.0×10⁻⁸ M) ofE₂ and E₁. In the present studies, the biphasic effect of E₂ reported byAmara and Dannies (Amara J F and Dannies P S (1983) Endocrinology 112,1141-1143) was not found. This may be explained by the differentconditions used to conduct the assays. The matter of assay cultureconditions with ER⁺ cells has been discussed (Zugmaier G et al. (1991) JSteroid Biochem Mol Biol 39, 681-685). Certainly however, the low E₂concentration for ED₅₀ still speaks to a problem with ERα as themediating receptor. Furthermore, the pattern reported in this Example isconsistent with physiological facts. Tumor formation by GH cells wasgreater in W/Fu rats treated with 25 mg estrogen pellets than inuntreated intact sexually mature females (Sorrentino J M et al. (1976) JNatl Cancer Inst 56, 1149-1154). Without a doubt, supraphysiologicallevels of estrogens were most effective in vivo. In contrast toestrogens, progesterone and cortisol had no effect on GH₄C₁ growth inculture FIG. 11. These steroids also did not promote GH cell tumorgrowth in vivo (Sorrentino J M et al. (1976) J Natl Cancer. Inst 56,1149-1154). The findings with androgens and GH₄C₁ cell growth shown inFIG. 11 revealed another important contribution made by the work in CDEserum supplemented cultures described herein. The Inventor had shownbefore that T promoted GH tumor growth in vivo (Sorrentino J M et al.(1976) J Natl Cancer Inst 56, 1149-1154). It was proposed at that timethat T was effective because it was metabolized to estrogens in the rat.Therefore, it was expected that T would be ineffective in culture. Theresults in FIG. 11 confirm this expectation. In this case, the newculture methods permitted resolution of an issue arising from previousin vivo observations. The dose-response results in FIG. 11 fortify aconclusion arrived at earlier that cell culture can be used to uncoverphysiologically important new information not accessible by in vitromethods (McKeehan W L et al. (1990) In Vitro Cell Dev Biol 26, 9-23).

Dose-Response Effects of Steroid Hormones with Hamster Kidney TumorCells in CDE Serum.

To explore the utility of the new culture conditions further, steroidhormone effects on the H301 Syrian hamster kidney tumor cells inD-MEM/F-12 containing 50% (v/v) CDE-horse serum were investigated. Thiscell line has two unique characteristics. First, tumors form from H301cells in Syrian hamsters only in response to exogenous estrogens(Sirbasku D A and Kirkland W L (1976) Endocrinology 98, 1260-1272). Itis very important to note that normal physiologic levels in intact adultfemale hamsters do not support tumor formation (Sirbasku D A andKirkland W L (1976) Endocrinology 98, 1260-1272). It is thought thatprogesterone from the normal estrus cycle suppresses growth in responseto physiological levels of estrogen (Kirkman H and Robbins M (1959) In:National Cancer Institute Monograph No. 1, National Institutes ofHealth, Bethesda, Md.). Second, these cells only form tumors in responseto estrogens. The other major classes of steroid hormones areineffective in vivo. The relative effectiveness of the three estrogenswith H301 cells was investigated (FIG. 12). Their potency was E₂>E₁>E₃.As with rat tumor cells, E₃ was markedly less effective than E₂ or E₁.E₂ and E₁ required 1.0×10⁻¹¹ M and 1.0×10⁻¹⁰ M, respectively, to achievesignificant (p<0.05) growth. The ED₅₀ concentration of E₂ is about 5 to9×10⁻¹¹ M. As expected from in vivo results (Sirbasku D A and Kirkland WL (1976) Endocrinology 98, 1260-1272), this concentration was higherthan for the rat pituitary tumor cells (FIG. 11) or rat mammary tumorcells (FIG. 3). In fact, they were as much as 100 to 1000-fold higherthan for human breast cancer cells (FIG. 10). In other tests shown inFIG. 12, progesterone, cortisol, T and DHT were all inactive. The higherestrogen concentrations required for significant growth of the H301cells in culture, coupled with the marked estrogen specificity as isseen in vivo (Sirbasku D A and Kirkland W L (1976) Endocrinology 98,1260-1272), indicate that the medium conditions used in this studyyielded physiologically germane results.

Dose-Response Effects of Steroid Hormones with Human Prostatic CarcinomaCells in CDE Serum.

In the final dose-response study, the potency of several classes ofsteroid hormones with the LNCaP cells was analyzed. This was done inD-MEM/F-12 containing 50% (v/v) CDE horse serum. Due to a point mutationwhich permits binding of both androgen and non-androgen hormones to theAR of LNCaP cells (Veldscholte J et al. (1990) Biochem Biophys ResCommun 173, 534-540; Veldscholte J et al. (1990) Biochim Biophys Acta1052, 187-194), the Inventor expected several classes of steroids topromote growth, albeit at concentrations compatible with their knownaffinities for the mutated receptor. This proved to be the case, asshown in FIG. 13. DHT and E₂ were the most potent steroids. In fact,they were equipotent. Both caused significant (p<0.05) growth at1.0×10⁻¹² M. Contrary to other reports (Schuurmans A L et al. (1988) TheProstate 12, 55-64; Sonnenschein C et al. (1989) Cancer Res 49,3474-3481; de Launoit Y et al. (1991) Cancer Res 51, 5165-5170; Lee C etal. (1995) Endocrinology 136, 796-803; Kim I et al. (1996) Endocrinology137, 991-999), the present study did not find that high concentrationsof DHT inhibited LNCaP growth. The potency of the steroid hormonestested was DHT=E₂>T>E₁>progesterone>E₃>cortisol. As potencies declined,saturation densities also decreased. The observed relative steroidpotencies agreed with those of others (Bélanger C et al. (1990) Ann NYAcad Sci 595, 399-402), and correlated with the expected binding of thevarious classes of steroids to the mutated AR of the LNCaP line.Additionally, the presently disclosed methods offered the advantage ofgreater growth responses. The results in FIG. 13 not only lend supportto the view that cultures containing a high concentration of CDE serumyield physiologically relevant information, but they also demonstratethat the new charcoal extraction method disclosed herein effectivelydepletes several classes of steroid hormones.

Discussion of Example 4

The methods presented in this Example show that mitogenic effects ofestrogens and androgens can now be measured at the picomoloar level.These highly sensitive assays can be used advantageously to assessclinical samples for inhibitor concentrations (after steroid depletion)of to establish that sufficient estrogens are present to cause growthpossibly in postmenopausal women. The concentrations that are measurablefall well below radioimmunoassay concentrations and will give anaccurate measure of the active estrogen (i.e. unbound) versus the totaldetermined by conventional procedures akin to radioimmunoassays. Theresults provided herein present a new approach to the question of whypostmenopausal women have sufficient levels of estrogens to promotebreast cancer cell growth. It is well known that ≧65% of the breastcancers in postmenopausal women are estrogen receptor positive. Theresults herein indicate that these cancers are so sensitive to estrogensthat even a reduced physiological concentration is sufficient to causegrowth. Breast cancer prevention by anti-hormone therapy must beevaluated on this new basis.

The results demonstrate clearly that serum contains at least oneestrogen reversible inhibitor and that it/they mediates physiologicallyrelevant sex steroid responses. The fact that CDE-horse serum iseffective with several cell lines of rodent and human origins indicatesthat the inhibitor or inhibitors are not species specific. Moreover, thefact that all of the ER⁺ cell lines responded similarly in these studiesto the different types of serum tested indicates that the inhibitor orinhibitors are ubiquitous in mammals. This suggests an importantphysiologic fact. Estrogen target tissue growth is coordinate in vivo.Administration of the hormone causes mitogenic effects in all of themajor target tissues such as breast, uterus, ovaries, female genitaltract, pituitary and specialized other tissues and cells. Therefore, thestudies presented imply that the inhibitor or inhibitors should beactive with several target tissues.

The results presented in this Example have special significance withregard to support for the conclusion that a new ERγ regulates growth. Inthese studies, growth is one-half maximally stimulated by 10-1,800 foldlower concentrations of E₂ than indicated by the Kd values expected ofthe classical ERα. According to the accepted theory of hormone binding,the K_(d) value represents the steroid concentration that one-halfsaturates the existing receptors. The following Table 4 summarizes theED₅₀ concentrations required for a one-half maximum growth in mediumcontaining 30 to 50% (v/v) CDE-serum versus the estrogen receptor K_(d)measured for the same or closely related cell lines. The new receptor isdiscussed further in a later Example.

TABLE 4 Comparisons of ED₅₀ and K_(d) as Evidence Supporting a New ERDesignated ERγ Fold-higher K_(d) Concentration ED₅₀ for E₂ Compared toED₅₀ for Cell Line Induced Growth K_(d) for E₂ Growth MTW9/PL2 1 × 10⁻¹²M  1.8 × 10⁻⁹ M 1.8 × 10³ T47D 1 × 10⁻¹² M 0.11 × 10⁻⁹ M 1.1 × 10³ GH₄C₁1 × 10⁻¹¹ M 0.25 × 10⁻⁹ M 25 H301 9 × 10⁻¹¹ M 0.87 × 10⁻⁹ M 10

Example 5 Thyroid Hormone Growth Effects in CDE-Horse Serum Prepared at34° C.

In this Example an assay system is described for testing substancesexpected to have thyroid hormone like activity. GH rat pituitary tumorcells are highly thyroid hormone responsive in serum-free defined medium(Eby J E et al. (1992) Anal Biochem 203, 317-325; Eby J E et al. (1992)J Cell Physiol 156, 588-600; Sato H et al. (1991) In Vitro Cell Dev Biol27A, 599-602). An example of this responsiveness with the GH₃ line isshown in FIG. 14. However, in serum-free defined medium, these cells arenot E₂ responsive when T₃ is omitted from the medium (FIG. 15). Duringevaluation of the role the GH cell lines in CDE-serum, it was found thatin D-MEM/F-12 with 2.5% (v/v) CDE-horse serum, T₃ caused substantialgrowth of the GH₄C₁, GH₁ and GH₃ rat pituitary tumor cell lines (FIG.16). However, at 50% (v/v) CDE-horse serum, only supraphysiologicconcentrations of thyroid hormone showed growth effects (FIG. 17).Nonetheless, the 34° C. CDE method described in the preceding Examplesis clearly functional to demonstrate both steroid hormone and thyroidhormone growth effects in culture. It is known that the thyroid hormonereceptor is a member of a superfamily of receptors that also includesthe steroid hormone receptors (Evans R M (1988) Science (Wash DC)240:889-895). Testing of substances expected to have thyroid hormonelike activity can be performed with the GH cell lines in the presence oflow concentrations of CDE-serum.

Discussion of Example 5

The removal of thyroid hormones from serum has been described beforeusing the Bio-Rad™ AG-1 X8 ion exchange resin (Samuels B H et al. (1979)Endocrinology 105, 80-85). Removal of T₃/T₄ by this method relies ontheir negative carboxylic acid charge at neutral pH. That method alsoremoves most of the other lower molecular weight charged substances fromserum. For some applications, this is not beneficial, particularly tothe demonstration of steroid hormone responsive cell growth in culture.Also, the ion exchange method does not remove the uncharged/hydrophobicsteroid hormones. Therefore, the AG-1 X8 method is more limited than the34° C. CDE method described herein.

Example 6 Estrogenic Effects in XAD-4™ Resin Treated Horse Serum

Horse serum depleted of steroid hormones by XAD-4™, prepared asdescribed in Example 2, was assayed to determine if it demonstratedestrogen reversible inhibition of ER⁺ cancer cell growth in culture.FIG. 18 shows the effects of XAD-4™ treated horse serum±10 nM E₂ withthe MTW9/PL2 cell line. Unmistakably, the pattern of cell response wasthe same as seen with CDE-horse serum. At 50% XAD-4™ serum (v/v), anestrogenic effect of 5.2 CPD was observed in 7 days. FIG. 19 shows asimilar experiment with T47D cells after 14 days. At 50% (v/v) XAD-4™treated serum, an estrogenic effect with T47D cells of 5.3 CPD wasobserved. The magnitudes of the estrogenic effects with both cell lineswere the same as observed with CDE-horse serum. Because both MTW9/PL2and T47D cells are sensitive to picomolar concentrations of estrogen, itwas evident that the XAD-4™ resin treatment effectively removed theendogenous sex steroids present in serum.

Discussion of Example 6

There is no previous report of the preparation steroid depleted serum bythis resin treatment method. As indicated in Example 2, the XAD-4™treatment method has particular applicability for the industrialpreparation of large volumes of steroid hormone depleted serum, and willallow the commercial supply of steroid depleted serum at reasonablecost. A preferred application for this steroid hormone stripped serum isin the biotechnology industry, in which cell culture is used to producemedically and otherwise commercially significant proteins and cellularproducts. Steroid hormone depleted serum has applicability beyond theER⁺ and AR⁺ cells described in this report. For example, hybridoma cellsare the sources of many important monoclonal antibodies. Depletion ofsteroids from the serum used to grow these cells will increase cellviability (e.g. cortisol is a potent cytotoxic agent for leukocyte celltypes), and therefore increase product yield. Moreover, steroid-strippedsera prepared in this way may stabilize hybridoma production ofdesirable immunoglobulins. The use of XAD™-4 extracted serum is alsoapplicable to development of hybridoma cells of medical significance andtherapeutic value. These and other applications of the XAD™-4 treatedserum for both commercial and diagnostic testing as well as forindustrial production of valuable cellular products are foreseen.

Example 7 Testing of Substances for Estrogenic Activity

The purported estrogenic effects of phenol red were tested and proven tobe unfounded. Further, the methods described in this Example exemplifymethods that are generally effective for assessing the steroidogenicactivity of any substance.

Examination of Phenol Red Indicator as an Estrogenic Substance.

The reported estrogenic action of phenol red and/or its lipophiliccontaminants has led to the widespread use of indicator free culturemedium to conduct endocrine studies in vitro (Berthois Y et al. (1986)Proc Natl Acad Sci USA 83, 2496-2500; Bindal R D et al. (1988) J SteroidBiochem 31, 287-293; Bindal R D and Katzenellenbogen J A (1988) J MedChem 31, 1978-1983). The generally accepted view is that the 8.1 mg/mL(i.e. about 23 μM) of phenol red present in the D-MEM/F-12 medium(Gibco-BRL) alone was sufficient to cause estrogenic effects. Despitethis, the results presented thus far in this disclosure show largemagnitude estrogen effects in D-MEM/F-12 tissue culture mediumcontaining the standard concentration of the indicator phenol red. Toensure that this potential problem was avoided in subsequent studies,the phenol red matter was further investigated, as reported(Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36,447-464). In so doing, nine estrogen receptor positive (ER⁺) cell linesrepresenting four target tissues and three species were selected. Phenolred was investigated using five different experimental protocols. First,E₂ responsive growth of all nine ER⁺ cells lines was compared in mediumwith and without the indicator. Second, using representative lines itwas determined whether phenol red was mitogenic in indicator freemedium. The dose-response effects of phenol red were compared directlyto those of E₂. Third, it investigated whether tamoxifen inhibitedgrowth equally in phenol red containing and indicator free medium. Thisstudy was based on a report indicating that antiestrogen effects shouldbe seen only in phenol red containing medium. Fourth, it wasinvestigated whether phenol red displaced the binding of ³H-E₂ using ER⁺intact human breast cancer cells. Fifth, it was investigated whether E₂and phenol red both acted as inducers of the progesterone receptor usinga human breast cancer cell line well known for this property (Horwitz KB and McGuire W L (1978) J Biol Chem 253, 2223-2228). All of theexperiments presented in this disclosure support the conclusion that theconcentration of phenol red contaminants in a standard culture mediumavailable today is not sufficient to cause estrogenic effects. Thestudies presented indicate that the real issue of how to demonstrateestrogenic effects in culture resides elsewhere than phenol red(Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36,447-464). It was found that demonstration of sex steroid hormonemitogenic effects in culture depends upon conditions that maximize theeffects of a serum-borne inhibitor(s). When the effects of the inhibitorare optimized, the presence or absence of phenol red makes no everydaydifference to the demonstration of estrogen mitogenic effects withseveral target cell types from diverse species (Moreno-Cuevas J E andSirbasku D A (2000) In Vitro Cell Dev Biol 36, 447-464).

Phenol Red Testing for Estrogenic Activity with MCF-7A Cells.

The original reports of the effect of phenol red or its impurities hadused the MCF-7 human breast cancer cells to assess estrogenic activity(Berthois Y et al. (1986) Proc Natl Acad Sci USA 83, 2496-2500; Bindal RD et al. (1988) J Steroid Biochem 31, 287-293; Bindal R D andKatzenellenbogen J A (1988) J Med Chem 31, 1978-1983). The initial studybegan with the MCF-7A strain of this population. As shown in FIG. 20A,growth was measured in the presence of increasing concentrations ofCDE-horse serum with and without phenol red in the medium and ±E₂.Concentrations of ≦10% (v/v) CDE-horse serum supported more than 5 CPD.Higher concentrations progressively inhibited in both indicatorcontaining and indicator free medium. In both types of medium, E₂ wasrequired to reverse the serum inhibition. To confirm that E₂ was equallyeffective in phenol red free and phenol red containing medium, theestrogenic effects shown in FIG. 20A were compared in both types ofmedium and at each serum concentration. The results of this analysis arepresented in FIG. 20B. The maximum estrogenic effect at 50% (v/v) serumwas 2.38 CPD (i.e. 2^(2.38) or 5.2-fold) in medium without indicator and2.56 CPD (i.e. 2^(2.56) or 5.9-fold) with phenol red. This differencewas not significant. Only at 5% (v/v) serum was there a significantly(p<0.05) greater estrogenic effect in phenol red free medium. However;in replicate experiments this <1.0 CPD effect was inconsistent. At allother serum concentrations, the growth differences between plus andminus phenol red were not significant.

Test of Phenol Red Effects with MCF-7K Cells.

The MCF-7K strain was routinely more estrogen responsive than the MCF-7Aline (Sirbasku D A and Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol36, 428-446). The MCF-7K cells also showed a serum concentrationdependent growth inhibition (FIG. 20C). The final degree of inhibitionat 50% (v/v) serum was independent of phenol red. Only in the presenceof 2.5, 5, 10 and 20% (v/v) CDE-horse serum were the estrogenic effectssignificantly greater in phenol red free (FIG. 20D). It is important tonote that while these differences were identified more often with theMCF-7K strain than the MCF-7A line, they were invariably small. Plainly,no serum concentration supported ≧1.0 CPD estrogenic effects in phenolred free medium compared to indicator free medium (FIG. 20D). In fact,deletion of phenol red improved estrogen responsiveness by an average ofonly 0.6 CPD with the MCF-7K line. When judged by the maximum estrogeniceffects achievable with MCF-7K cells in 50% (v/v) CDE-horse serum, plusand minus phenol red gave indistinguishable results of CPD 3.01(8.0-fold) and CPD 2.99 (7.9-fold), respectively (FIG. 20D).

Phenol Red Testing for Estrogenic Activity with T47D and ZR-75-1 Cells.

The same experiments just described above with the MCF-7 cell strainswere repeated with T47D and ZR-75-1 cells. These lines weresubstantially more estrogen stimulated in CDE-serum than MCF-7 cells(Sirbasku D A and Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36,428-446) and hence were expected to be more sensitive to phenolred/contaminants.

Phenol Red and T47D Cells.

T47D cells were grown in medium with CDE-horse serum both with andwithout phenol red (FIG. 21A). Low concentrations of serum (i.e. ≦2%)promoted growth. Higher concentrations progressively inhibited growthirrespective of indicator content. In both media, E₂ was required toreverse the inhibition (FIG. 21A). In 50% (v/v) CDE-horse serum, themaximum E₂ responses were 2^(5.35) (41-fold) and 2^(5.29) (39-fold) inphenol red containing and indicator free medium, respectively (FIG.21B). Only at low serum concentrations were phenol red effects observedin any experiment. In some replicates, the phenol red effect wasopposite to that expected. For example, in the experiment shown in FIG.21B, 0.5 to 2.5% serum showed significantly (p<0.05) greater estrogeniceffects in the presence of phenol red. These results graphicallyillustrate the hazards of interpreting 1.0 CPD responses either in favorof phenol red/contaminants as estrogens or in opposition to thisproposal.

Phenol Red and ZR-75-1 Cells.

ZR-75-1 cells showed similar results as the T47D line. Serum caused aninhibition of growth that was undoubtedly unrelated to phenol red (FIG.21C). In both types of medium, and at every serum concentration tested,E₂ was required to reverse the inhibition (FIG. 21C). In 50% (v/v)serum, ZR-75-1 cells showed maximum estrogenic effects of 2^(3.39)(10.5-fold) and 2^(3.49) (11.2-fold) in medium with and withoutindicator, respectively (FIG. 21D). As seen with T47D cells, the ZR-75-1line showed greater estrogenic effects in medium with phenol red than inmedium without indicator when the serum was 0.5, 5 or 10% (v/v) (FIG.21D).

Phenol Red Testing for Estrogenic Activity with MTW9/PL2 Cells.

The next experiments were done with MTW9/PL2 rat mammary tumor cells(FIG. 22A). They were inhibited by high concentrations of CDE-horseserum with and without indicator. E₂ was required to reverse theinhibition in both types of medium (FIG. 22A). The maximum estrogeniceffects in 50% serum were 2^(5.82) (56-fold) and 2^(5.69) (52-fold) withand without phenol red, respectively (FIG. 22B). In the experiment shownin FIG. 22B, estrogenic effects were unpredictably greater in phenol redfree medium than in medium with indicator. This was observed at lowserum concentrations (i.e. 0.5 and 1.0%) and again at higher levels(i.e. 20 and 30%). Although suggesting a phenol red effect, theseresults in fact only serve to emphasize the pitfalls of accepting smallchanges as meaningful even though they are significant at p<0.05. Whenestrogenic effects were found with MTW9/PL2 cells in phenol red freeconditions, they invariably were ≦1.0 CPD. The sum of the studies withMTW9/PL2 cells did not yield a predictable correlation betweenestrogenic effects in the absence of the indicator and serumconcentrations.

Other Cell Lines Tested for Growth±Phenol Red and ±E₂.

The results presented above were replicated with the GH₁ and GH₄C₁ ratpituitary tumor cell lines as well as with the H301 cells and the LNCaPcell line (Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell DevBiol 36, 447-464). Again, the presence or absence of the indicator inthe medium containing CDE-horse serum had no effect whatever on thedemonstration of the usual high estrogenic effects with these cells.

Direct Test of Phenol Red Estrogenic Activity.

Three cell lines were selected for a direct test of phenol red as amitogen. The MCF-7A line was used because it most closely approximatedthe origin and passage age of the cells used to conduct the originalstudy of phenol red as a weak estrogen (Berthois Y et al. (1986) ProcNatl Acad Sci USA 83, 2496-2500). The T47D cells were chosen becausethey are the most estrogen responsive human breast cancer cell lineavailable today (Sirbasku D A and Moreno-Cuevas J E (2000) In Vitro CellDev Biol 36, 428-446). The MTW9/PL2 cells were chosen as an example of ahighly estrogen responsive rodent origin line (Moreno-Cuevas J E andSirbasku D A (2000) In Vitro Cell Dev Biol 36, 410-427; Sirbasku D A andMoreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446). The assayswere done in phenol red free D-MEM/F-12 supplemented with 30% CDE-HS.This concentration was chosen even though it is not as inhibitory as 50%(v/v) serum. This selection was made to reduce possible interactions ofthe phenol red/contaminant with serum proteins while still retaining asignificant inhibitory effect. Phenol red concentrations of up to 16mg/L were added to this medium. This highest level was twice that instandard, commercially formulated Gibco-BRL D-MEM/F-12. Severaldifferent manufacturing lots of aqueous phenol red gave equivalentresults. The preparations used in this study ranged in age from newlyobtained to more than ten year old laboratory stocks. These experimentsgave unmistakable results. There was no increase in the growth of any ofthe cell lines in response to phenol red (FIG. 23A). By comparison,parallel cultures receiving E₂ showed sizable 2 to 5 CPD responses tothe natural hormone (FIG. 23B). E₂ at 1.0×10⁻¹⁰ M optimized growth ofall three cell lines. The ED₅₀ concentrations of E₂ were 3.0×10⁻¹² M.Significant (p<0.05) estrogenic effects were observed at 1.0×10⁻¹² M.The results presented in FIG. 23 indicate that the culture conditionsused in this study could reasonably be expected to detect responses dueto contaminants present at the concentrations indicated in the originalreports (Berthois Y et al. (1986) Proc Natl Acad Sci USA 83, 2496-2500;Bindal R D et al. (1988) J Steroid Biochem 31, 287-293; Bindal R D andKatzenellenbogen J A (1988) J Med Chem 31, 1978-1983).

Comparison of E₂ Potency in Medium with and without Phenol Red.

As described above in Table 4, the T47D and MTW9/PL2 cells growsignificantly in response to 1.0×10⁻¹² M E₂. The D-MEM/F-12 used inthose studies also contained about 23 μM phenol red. When the results ofthose studies were compared to the experiments in FIG. 23B, done inD-MEM/F-12 without indicator, the estrogen dose response curves werevery similar. The conclusion is straightforward. E₂ dose-responses werenot affected by phenol red. If phenol red lipophilic contaminants werepresent at the concentrations originally suggested (Berthois Y et al.(1986) Proc Natl Acad Sci USA 83, 2496-2500; Bindal R D et al. (1988) JSteroid Biochem 31, 287-293; Bindal R D and Katzenellenbogen J A (1988)J Med Chem 31, 1978-1983) they should have masked the observation ofpicomolar effects of exogenous estrogens.

Effect of Phenol Red on Binding of ³H-E₂ to Intact Cells.

For the next study, intact T47D cells were used to measure the effectsof phenol red on estrogen receptor binding. The cells were incubatedwith 5 nM ³H-E₂ and the effects of addition of increasing concentrationsof unlabeled E₂ assessed (Table 5). A 100-fold excess of unlabeled E₂displaced 75% of the binding of ³H-E₂. By this criterion, 75% of thebinding of ³H-E₂ was specific to estrogen receptors (Chamness G C andMcGuire W L (1975) Steroids 26, 538-542). The same analysis wasconducted with aqueous preparations of phenol red. Even at 16 mg/L, theindicator did not reduce the binding of ³H-E₂ (Table 5). This was trueno matter which batch of indicator was analyzed (results not shown). Thephenol red used for the experiment shown in Table 5 was approximatelythe same age (purchased in 1986) as the date of the original report(Berthois Y et al. (1986) Proc Natl Acad Sci USA 83, 2496-2500). Theseresults raise the question how often preparations of phenol redpurchased at that time as an aqueous membrane filtered product containeda sufficient level of contaminants to elicit an estrogenic effect.

TABLE 5 DISPLACEMENT OF ³H-E₂ BINDING TO INTACT T47D CELLS BY UNLABELEDE₂ OR UNLABELED PHENOL RED IN INDICATOR FREE AND SERUM-FREE D-MEM/F-12FOR TWO HOURS AT 37° C. Control - No Additions 12,458 ± 1615 100% (5 nM³H-E₂ only) 2.5 nM Unlabeled E₂ 12,177 ± 872 98% 5.0 nM Unlabeled E₂ 8,756 ± 588 70% 50 nM Unlabeled E₂  7,898 ± 744 63% 250 nM Unlabeled E₂ 4,892 ± 194 39% 500 nM Unlabeled E₂  3,494 ± 127 28% 1000 nM UnlabeledE₂  2,543 ± 304 20% 1 mg/L Phenol Red 12,670 ± 727 102% 2 mg/L PhenolRed 13,874 ± 906 111% 4 mg/L Phenol Red 11,730 ± 566 94% 8 mg/L PhenolRed 12,357 ± 664 99% 16 mg/L Phenol Red 13,748 ± 998 110%

Comparison of the E₂ and Phenol Red Induction of Progesterone Receptors.

Another putative function of phenol red was to induce progesteronereceptors in estrogen sensitive cells. An investigation was made as towhether the indicator induced an increase in the progesterone receptorsof T47D cells which contain these sites (Horwitz K B et al. (1978)Cancer Res 38, 2434-2437). In a first study, the kinetics ofprogesterone receptor induction versus estrogen concentration in phenolred free medium were investigated (FIG. 24A). E₂ levels as low as1.0×10⁻¹² M caused a significant two-fold increase in receptor contentin four days. At 1.0×10⁻⁸ M, E₂ induced a four-fold increase inprogesterone receptors in four days. Clearly, E₂ induced a time andconcentration dependent increase in the progesterone receptors with T47Dcells. Next, this same analysis was done with phenol red over aconcentration range of 1 to 16 mg/L (FIG. 24B). Phenol red induced asmall increase in progesterone receptors at 8 and 16 mg/L after fourdays. This induction was about the same as caused by 1.0×10⁻¹⁴ M E₂(FIG. 24A). These results indicate that if estrogenic contaminants arepresent in phenol red, they are most likely in the 10⁻¹⁴ M range evenassuming equal receptor binding capacity to E₂. This point is importantbecause the active agent is thought to be only a trace impurity in manybatches of phenol red (Bindal R D et al. (1988) J Med Chem 31,1978-1983). The impurities bind to the estrogen receptor with only 50%of the affinity of E₂. The impurity was expected to be 0.002% of thephenol red concentration. Based on test results that employed manydifferent batches of Gibco-BRL D-MEM/F-12, this concentration of theimpurity seems highly unlikely in the medium commercially availabletoday.

Discussion of Example 7

The studies of the effects of phenol red or its lipophilic impuritiesdemonstrate the usefulness of the presently disclosed methods for theassessment of estrogenic and androgenic activity of commerciallyprepared materials, substances present or extracted from environmentalor food sources or other sources that are thought to contain suchactivities. The testing can be approached by three separate methods, asshown by examples with phenol red. (1) Compounds or other preparationsand substances can be tested for growth activity with human or rodentcell lines depending upon the information sought. Potency can beestablished as UNITS based on E₂ or any other estrogen or androgenrequired. This permits direct expression of the estrogen like activityor androgen like activity per volume or mass of the substance underevaluation. Levels can be measured without regard for expensivedevelopment of a radio immunoassay that in the end still does not yieldevidence of biological activity as a sex steroid hormone analog (agonistor antagonist). The use of rodent cell lines opens the possibility ofdirect comparison to in vivo activity if required. The effects ofhormone-like substances can be tested with human cell lines in athymicnude mice or SCID mice as required. (2) Another form of analysis isdirect measure of potency by ³H-E₂ or ³H-DHT binding displacementanalysis from whole cells or extracted estrogen receptors. An examplewith ³H-E₂ and whole cells is shown in Table 5. The two differentbinding assays offer different information. Whole cells have apredominance of hydrophobic sites (i.e. membranes) that absorblipophilic substances and therefore may attenuate their activity. Use ofcell extracted sex steroid hormone receptors permits direct measure ofthe potential of a substance to act as a hormone independent of itsbiological effects. (3) Finally, use of the progesterone receptoranalysis permits evaluation of substances and preparations by a methodentirely independent of growth. This is a gene expression based analysisthat permits evaluation that can be used to supplement growth data or beused in place of growth analysis. The MTW9/PL2 cells have been shownabove to be suitable for this purpose.

Example 8 Testing of Substances for Inhibitor-Like Activity

In studies described in this Example, TGFα, TGFβ1, EGF, IGF-I, IGF-IIand insulin were tested in the cell growth assay described in thepreceding Examples, substituting those proteins for the serum-borneinhibitor contained in the preferred CDE serum.

TGFβ1 as a Substitute for the Serum-Borne Estrogen Reversible Inhibitor.

Normal mouse mammary (Silberstein G B and Daniel C W (1987) Science(Wash DC) 237, 291-293; Silberstein G B et al. (1992) Dev Biol 152,354-362) and normal human breast epithelial cell growth is inhibited byTGFβ (Bronzert D A et al. (1990) Mol Endocrinol 4, 981-989).Additionally, human breast cancer cells are inhibited by TGFβ (Knabbe Cet al. (1987) Cell 48, 417-428; Arteaga C L et al. (1988) Cancer Res 48,3898-3904; Arteaga C L et al. (1990) Cell Growth Diff 1, 367-374). TGFβalso inhibits the GH₄C₁ rat pituitary tumor cells (Ramsdell J S (1991)Endocrinology 128, 1981-1990) and the LNCaP human prostatic carcinomacells (Schuurmans A L et al. (1988) The Prostate 12, 55-64; Wilding G etal. (1989) Mol Cell Endocrinol 62, 79-87; Carruba G et al (1994)Steroids 59, 412-420; Castagnetta L A and Carruba G (1995) Ciba FoundSymp 191, 269-286; Kim I Y et al. (1996) Endocrinology 137, 991-999). Instudies presented next, replacement of the serum-borne inhibitor withTGFβ was attempted. A number of related forms of this inhibitor areknown (Clark D A and Coker R (1998) Int J Biochem Cell Biol 30, 293-298;Massagué J (1998) Annu Rev Biochem 67, 753-791). TGFβ1 and TGFβ2 aremost often studied and commonly have similar potencies. For example,they are equipotent with human breast cancers cells (Zugmaier G et al.(1989) J Cell Physol 141, 353-361). TGFβ1 was chosen for the instantstudy. Without a doubt, a number of the key cell lines used throughoutthe Examples were inhibited by TGFβ. It was therefore consideredessential to ask if TGFβ was the estrogen reversible inhibitor.

TGFβ1 and MCF-7 Cells.

Because MCF-7 cells are probably the most studied human breast cancerline today, this next work began with those cells. TGFβ has beendescribed as a hormone regulated autocrine inhibitor of the ER⁺ MCF-7human breast cancer cell growth (Knabbe C et al. (1987) Cell 48,417-428). In the present study, to test if TGFβ1 substituted for theserum-borne inhibitor with these cells, they were grown in D-MEM/F-12containing 2.5% (v/v) CDE-horse serum plus increasing concentrations oftransforming growth factor and ±E₂. The results in FIG. 25A show thateven 50 ng/mL of TGFβ1 caused only a modest inhibition of MCF-7K cellgrowth. Cell numbers were reduced from 350,000 to 200,000 per dish. Thisdifference was significant (p<0.05). Nevertheless, the estrogen reversalof the inhibition was no larger than the E₂ effect observed inD-MEM/F-12 containing 2.5% (v/v) horse serum without TGFβ1 FIG. 25A.Furthermore, when the cell number data were expressed as CPD (insertFIG. 25A), it was definite that TGFβ1 was at best a very modestinhibitor and that there was no TGFβ1 related estrogenic effect.

TGFβ1 and MTW9/PL2 Cells.

The next study was performed because the MTW9/PL2 cells are the onlyknown estrogen growth responsive rat cell line derived from a hormoneresponsive carcinogen induced tumor. A similar analysis was done withthe MTW9/PL2 rat mammary tumor cells (FIG. 25B). TGFβ1 reduced cellnumbers from 350,000 to 100,000 per dish. This was significant (p<0.05).However, the presentation of cell number results only tends toexaggerate the effects of TGFβ1. When the results were converted to CPD(insert FIG. 25B), the actual inhibition was 1.5 CPD. This was at most a25% decrease in growth rate. As shown, there was no estrogen reversal ofthe TGFβ1 inhibition with MTW9/PL2 cells.

TGFβ1 and Other ER⁺ Cell Lines.

The effects of TGFβ1 at 50 ng/mL±E₂ were also investigated with theother cell lines used in this study. The MCF-7A, T47D and ZR-75-1 humanbreast cancer cells were inhibited by TGFβ1 (FIG. 26A). From theseresults, and those in FIG. 25A, it was clear that the MCF-7 cells werethe most sensitive of the ER⁺ human breast cancer lines tested.Irrespective of the line, E₂ had no influence on the TGFβ1 mediatedinhibitions (FIG. 26A). The same experiments were done with the LNCaPcells and the GH₄C₁ pituitary line (FIG. 26A). They were more sensitiveto TGFβ1 than breast cancer cells. Nonetheless, the TGFβ1 effects werenot reversed by E₂. When the cell number decreases presented in FIG. 26Awere converted to CPD, it was clear that the TGFβ1 effects werenegligible and that E₂ was of no significant consequence (FIG. 26B).Thus, TGFβ1 did not substitute for the estrogen reversible inhibitor(s)in CDE serum with any of the sex steroid sensitive ER⁺ cell linestested.

TGFα and EGF as Substitutes for the Estrogen Reversible Inhibitor in CDESerum.

The EGF family of mitogens and receptors has been linked to breastcancer proliferation, invasion and progression (Dickson R B and LippmanM E (1987) Endocr Rev 8, 29-43; Norman no N et al. (1994) Breast CancerRes Treat 29, 11-27; Ethier S P (1995) J Natl Cancer Inst 87, 964-973;de Jung J S et al. (1998) J Pathol 184, 44-52 and 53-57). Most prominentamong these polypeptide mitogens has been the EGF analogue, TGFα(Dickson R B and Lippman M E (1987) Endocr Rev 8, 29-43; de Jung J S etal. (1998) J Pathol 184, 44-52 and 53-57). Estrogen induced secretion ofTGFα is thought to create an autocrine loop that promotes breast cancercell growth (Dickson R B et al. (1985) Endocrinology 118, 138-142;Dickson R B et al. (1986) Cancer Res 46, 1707-1713; Dickson R B et al.(1986) Science (Wash DC) 232, 1542-1543; Dickson R B and Lippman M E(1987) Endocr Rev 8, 29-43; Derrick R (1988) Cell 54, 593-595; Arrack BA et al. (1990) Cancer Res 50, 299-303; Kenney N J et al. (1993) J CellPhysiol 156, 497-514; Normanno N et al. (1994) Breast Cancer Res Treat29, 11-27; Dickson R B et al. (1987) Proc Natl Acad Sci USA 84, 837-841;Salomon D S et al. (1984) Cancer Res 44, 4069-4077; Liu S C et al.(1987) Mol Endocrinol 1, 683-692). TGFα is also thought to potentiateestrogen action in uterus (Nelson K G et al. (1992) Endocrinology 131,1657-1664) as well as to regulate the EGF receptor in this tissue(DiAugustine R P et al. (1988) Endocrinology 122, 2355-2363; Huet-HudsonY M et al. (1990) Mol Endocrinol 4, 510-523; Mukku V R and Stancel G M(1985) J Biol Chem 260, 9820-9824). The culture conditions describedherein offer a new opportunity to test the autocrine growth model underconditions not previously available. Application of the new cell growthassays allowed a direct test to determine if an autocrine/intacrinegrowth factor loop explains the estrogen reversal of the seruminhibition.

EGF and TGFα as Substitutes for E₂.

Growth of the MCF-7A, MCF-7K, T47D and ZR-75-1 cells was measured inD-MEM/F-12 containing increasing concentrations of CDE horse serum withand without exogenous EGF or TGFα. The results with the four cell linesare shown in FIGS. 27A, 27B, 27C, and 27D, respectively. As expected,CDE horse serum was progressively inhibitory at concentrations >5%(v/v). The addition of growth saturating concentrations (Karey K P andSirbasku D A (1988) Cancer Res 48, 4083-4092) of EGF or TGFα did notreverse the effects of the serum-borne inhibitor. In control cultureswithout added polypeptide mitogens, E₂ completely reversed the seruminhibition. These results again confirm the same conclusion arrived atearlier using an entirely different approach (Karey K P and Sirbasku D A(1988) Cancer Res 48, 4083-4092). Direct evidence for obligatoryEGF/TGFα autocrine loops in estrogen responsive cell growth simply hasnot yet been established. In fact, there is solid in vivo evidence thatchallenges an EGF/TGFα autocrine loop as active in the action ofestrogens (Arteaga C L et al. (1988) Mol Endocrinol 2, 1064-1069).

IGF-I, IGF-II and Insulin as Substitutes for Estrogen Action.

Insulin-like growth factors I and II (IGF-I and IGF-II) promote breastcancer cell growth (Furlanetto R W and DiCarlo J N (1984) Cancer Res 44,2122-2128; Myal Y et al. (1984) Cancer Res 44, 5486-5490; Dickson R Band Lippman M E (1987) Endocr Rev 8, 29-43; Karey K P and Sirbasku D A(1988) Cancer Res 48, 4083-4092; Ogasawara M and Sirbasku D A (1988) InVitro Cell Dev Biol 24, 911-920; Stewart A J et al. (1990) J Biol Chem265, 2172-2178). IGF-I related proteins (Huff K K et al. (1986) CancerRes 46, 4613-4619; Huff K K et al. (1988) Mol Endocrinol 2, 200-208;Dickson R B and Lippman M E (1987) Endocr Rev 8, 29-43; Minute F et al.(1987) Mol Cell Endocrinol 54, 17-184, as well. IGF-II (Yee D et al.(1988) Cancer Res 48, 6691-6696; Osborne C K et al. (1989) MolEndocrinol 3, 1701-1709), are thought of as possible autocrine/paracrinemitogens. Their secretion in response to hormones has been proposed(Dickson R B and Lippman M E (1987) Endocr Rev 8, 29-43; Huff K K et al.(1988) Mol Endocrinol 2, 200-208; Osborne C K et al. (1989) MolEndocrinol 3, 1701-1709). Insulin itself is likely an endocrinemediator. In the instant study, it was investigated whether exogenousIGF-I addition to cultures containing CDE-horse serum substituted forthe inhibition reversing effects of estrogens with human breast cancercells. FIG. 28A and FIG. 28B show the results with the MCF-7K and MCF-7Acells, respectively. Clearly, 1.0 μg/mL IGF-I did not reverse the seruminhibition. This was true despite the fact that this concentration ofadded IGF-I was much more than growth saturating (Karey K P and SirbaskuD A (1988) Cancer Res 48, 4083-4092). Duplicate studies with the T47Dcells gave the same results (FIG. 28C). It should be noted that IGF-I isactive with breast cancer cells even in the presence of serum(Furlanetto R W and DiCarlo J N (1984) Cancer Res 44, 2122-2128; Myal Yet al. (1984) Cancer Res 44, 5486-5490; Osborne C K et al. (1989) MolEndocrinol 3, 1701-1709; Stewart A J et al. (1990) J Biol Chem 265,2172-2178; Cullen K J et al. (1990) Cancer Res 53, 48-53) that containsspecific growth factor binding proteins (Rechler M et al. (1980)Endocrinology 107, 1451-1459). Human breast cancer cells also secretebinding proteins for the insulin-like growth factors (Yee D et al.(1991) Breast Cancer Treat Res 18, 3-10). Binding of the insulin-likefactors to carrier proteins may attenuate activity (Zapf J et al. (1978)J Clin Invest 63, 1077-1084), have both inhibiting and activatingeffects (De Mellow J S et al. (1988) Biochem Biophys Res Commun 156,199-204), or enhance biological action (Elgin R et al. (1987) Proc NatlAcad Sci USA 84, 3254-3258; Blum W F et al. (1989) Endocrinology 125,766-772). In parallel studies (data not shown), the effects of IGF-IIwere assayed with the same breast cancer lines under the conditions usedwith IGF-I. Even at 500 ng/mL, IGF-II did not reverse the inhibitoryeffects of 10 to 50% (v/v) CDE serum. In another related test, insulinat 10 ng/mL to 10 μg/mL did not reverse the inhibition caused by 50%(v/v) CDE serum. The results with insulin, IGF-I and IGF-II weremutually supportive because these mitogens promote growth via a commonreceptor (Rechler M et al. (1980) Endocrinology 107, 1451-1459; Karey KP and Sirbasku D A (1988) Cancer Res 48, 4083-4092; Osborne C K et al.(1989) Mol Endocrinol 3, 1701-1709; Stewart A J et al. (1990) J BiolChem 265, 2172-2178). The insulin results were also important in anotherway. This hormone does not interact with binding proteins and hencetheir presence in medium will not influence insulin action. Theseresults again confirm the same conclusion arrived at earlier using anentirely different approach (Karey K P and Sirbasku D A (1988) CancerRes 48, 4083-4092). Direct evidence for obligatory IGF-1/IGF-IIautocrine loops in estrogen responsive cell growth simply has not beenconfirmed yet. In fact, there is solid in vivo evidence to the challengeIGF-1/IGF-II autocrine loop participation in the action of estrogens(Arteaga C L et al. (1989) J Clin Invest 84, 1418-1423).

Discussion of Example 8

From this series of experiments, it can be readily appreciated that anyother natural or synthetic protein or other substance can be similarlytested for cancer cell growth inhibiting activity akin to theserum-derived inhibitor in the CDE horse serum. Also, the same XAD™-4and CDE extraction protocols may also be applied to body fluids andsecretions other than serum, and the extracted fluids may be assayed asdescribed for inhibitor activity. Such fluids or secretions includeplasma, urine, seminal fluid, milk, colostrum and mucus. An XAD™-4column is especially suited for preparing a steroid hormone depletedspecimen from a small sample of body fluid.

Conceptual Derivations from this Study.

These results also have a direct bearing on a number of hypothesesadvanced to explain how estrogens cause target tissue cell growth. Thedevelopment of the new methods herein provided a unique opportunity toreevaluate the most widely cited proposals under consideration. It wasconcluded that serum contains an inhibitor that effectively blocks ER⁺and AR⁺ cell growth. Furthermore, physiologic concentrations of sexsteroid hormones reverse this inhibition. The results were uniformly thesame no matter from which species the cell lines were derived or whichspecies was the source of the serum. In every case, the effects of thevarious classes of steroid hormones on the different cell lines wereconsistent with their known tumor forming/growth properties in vivo orpublished responses in vitro. These results provide new insights intothe following proposed mechanisms.

Serum Factor Regulation—Demonstration of Estrogen Responsiveness.

The literature describing positive sex steroid hormone growth effects isnotably weighted in favor of the use of serum-supplemented cultures. Infact, a review made of the literature (Briand P and Lykkesfeldt A E(1986) Anticancer Res 6, 85-90; Wiese T E et al. (1992) In Vitro CellDev Biol 28A, 595-602) indicates that most past studies have used mediumcontaining ≦20% (v/v) steroid hormone depleted serum. Although otherinvestigators have reported estrogenic effects in “serum-free definedculture”, these studies actually used conditions that included aprolonged preincubation in the presence of serum (Allegra J C andLippman M E (1978) Cancer Res 38, 3823-3829; Briand P and Lykkesfeldt AE (1986) Anticancer Res 6, 85-90; Darbre P D et al. (1984) Cancer Res44, 2790-2793). The results presented in preceding Examples demonstrateclearly that large magnitude effects are readily demonstrable in mediumwith CDE-serum and that as the CDE-serum concentrations increase to amaximum useable level of 50%, cell growth is inhibited and estrogensinvariably reverse these effects. In light of those results, it wasclear that the presence of serum, or a factor(s) contained in serum,made possible the demonstration of sex hormone dependent growth inculture.

The Endocrine Estromedin Hypothesis—Positive Indirect Control.

In 1978 it was proposed (Sirbasku D A (1978) Proc Natl Acad Sci USA 75,3786-3790) that growth of estrogen target tissues was not mediateddirectly by these hormones, but was instead controlled indirectly bysteroid inducible circulating growth factors (i.e. endocrineestromedins). Estromedins were proposed to be secreted by target tissuessuch as uterus, kidney and pituitary, and to act in concert tosimultaneously promote the growth of all ER⁺ target tissues (Sirbasku DA (1978) Proc Natl Acad Sci USA 75, 3786-3790; Sirbasku D A (1981)Banbury Report 8, 425-443; Ikeda T et al. (1982) In Vitro 18, 961-979).The estromedin hypothesis arose from the observation that reproduciblein vitro direct estrogen mitogenic effects were not identifiable(Sirbasku D A (1978) Proc Natl Acad Sci USA 75, 3786-3790; Sirbasku D A(1981) Banbury Report 8, 425-443; Ikeda T et al. (1982) In Vitro 18,961-979). It must be emphasized that the original estromedin hypothesisrested entirely upon the failure to demonstrate large magnitude estrogenmitogenic effects in culture with cell lines confirmed to form steroidhormone responsive tumors in host animals. When estrogen effects wereclearly observed with the MTW9/PL2 rat mammary tumor cells in culture,as described herein and reported (Moreno-Cuevas J E and Sirbasku D A(2000) In Vitro Cell Dev Biol 36, 410-427; Sirbasku D A andMoreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446), it wasapparent that the endocrine estromedin model required furtherevaluation. It was reasoned that extension of these results toadditional ER⁺ cell lines, including those from other species anddiverse target tissues, would either provide important support for theearlier hypothesis or disprove it. In the work disclosed herein, thisreassessment has been accomplished. All of the ER⁺ cells tested, as wellas one androgen sensitive AR⁺ human cancer line, manifested substantialgrowth in response to the appropriate steroid hormones in culturescontaining inhibiting concentrations of CDE serum. There can be no doubtthat steroid hormones act positively to promote target tumor cellgrowth. The results presented in this report plainly nullify theprevious endocrine estromedin model of steroid hormone responsive cellgrowth. The disapproval of the earlier endocrine estromedin modelreopened the question of how estrogens and other factors regulate sexsteroid responsive growth.

The Autocrine and Paracrine Models—Positive Indirect Control.

In the studies described in this Example, it was investigated whetherexogenous growth factors mimic the inhibitor reversing effects ofestrogens. The EGF/TGFα and insulin-like families were focused onbecause of their high biological potencies and physiologic relevance.These growth factors were expected to substitute for steroid hormonesbased on the autocrine loop mechanisms proposed earlier. Despite thisexpectation, polypeptide growth factors did not substitute for theestrogens. They were inactive in the presence of the serum-borneinhibitor. In point of fact, deduction indicates that it makes nopractical difference whether the growth factors were autocrine orparacrine in origin. The presence of the serum inhibitor in effectblocks all mitogenic action except that exerted by the steroid hormones.This is a preferred feature of the serum-borne inhibitor(s) disclosedherein, and is further described in Examples which follow, when the useof serum-free defined culture is described. These results also indicatethat the search for the regulatory mechanism controlling estrogendependent growth must seek new directions. Since the estrogenic effectsseen in CDE-serum are the largest yet recorded, CDE is the preferredsource of the regulator in the cell growth assays.

Culture Parallels In Vivo Growth Regulation.

The results shown in this Example have another important implication.Usually normal in vivo tissues are bathed in growth factor containingfluids. Mitogens within tissues may be of local origin or may be derivedfrom the circulation (Gospodarowitz D and Moran J S (1976) Annu RevBiochem 45, 531-558; Goustin A S et al. (1986) Cancer Res 46,1015-1029). If growth factors have unrestricted freedom to stimulatecell proliferation, normal formation and architecture of the tissueswould not develop nor could they be maintained. Manifestly, tissuearchitecture would be disrupted. In fact, this is one definition ofcancer (Sonnenschein C and Soto A M (2000)Molecular Carcinogenesis 29,205-211). The properties of a serum-borne inhibitor that counterbalancesunrestricted growth merit serious further consideration with regard tohow cancers develop in steroid hormone sensitive tissues. Othersresearchers have also arrived at this conclusion (Soto A M andSonnenschein C (1985) J Steroid Biochem 23, 87-94).

The Estrocolyone Hypothesis—Negative Indirect Regulation.

The estrocolyone model (Soto A M and Sonnenschein C (1987) Endocr Rev 8,44-52) is an indirect negative mechanism based on regulation of sexsteroid hormone dependent cells via a serum-borne inhibitor. Theinhibitor blocks growth promoted by non-steroidal mitogens such asgrowth factors and diferric transferrin. Sonnenschein and Soto firstproposed that estrocolyone acted at the cell surface via specificreceptors. The effects of sex steroid hormones were to bind estrocolyoneand prevent it from associating with the cells. Only low physiologicconcentrations of sex steroid hormones were needed for this function.The special emphasis of this model was that sex steroid hormones did notact through intracellular located DNA binding receptors (i.e. cytosolicor nuclear sites). These intracellular sites had no growth function.Hence, this was an indirect negative mechanism (Soto A M andSonnenschein C (1987) Endocr Rev 8, 44-52). The results presented inthis disclosure are in agreement with the serum-borne mediator aspect ofthe estrocolyone hypothesis. There is no doubt that serum from severalspecies contains a steroid hormone reversible inhibitor and that itsisolation and molecular characterization will be a major advance withboth practical and conceptual applications. With regard to the actionsite of the steroid hormones, these results differ from the estrocolyonehypothesis as described (Soto A M and Sonnenschein C (1987) Endocr Rev8, 44-52). The tentative identification of several estrocolyonecandidates have been described, and in U.S. Pat. Nos. 4,859,585(Sonnenschein) and 5,135,849 (Soto), the issue of properties was raisedagain, but with different conclusions than published earlier.

The Positive Direct Model—Steroid Hormone Receptor Mediation.

The one mechanism most widely accepted regarding steroid hormones andgrowth involves the nuclear located DNA binding ERα receptor (Gorski Jand Hansen J C (1987) Steroids 49, 461-475). Growth is thought to bemediation by specific cytosolic and/or nuclear located receptors thatultimately alter DNA transcription to regulate gene activity. Resultsfrom many laboratories support this mechanism (Jensen E V and Jacobson HI (1962) Recent Prog Horm Res 18, 387-414; Gorski J et al. (1968) RecentProg Horm Res 24, 45-80; Jensen E V et al. (1968) Proc Natl Acad Sci USA59, 632-638; Jensen E V and DeSombre E R (1973) Science (Wash DC) 182,126-134; Anderson J N et al. (1974) Endocrinology 95, 174-178; O'MalleyB W and Means A R (1974) Science (Wash DC) 183, 610-620; Lippman M E(1977) Cancer Res 37, 1901-1907; Harris J and Gorski J (1978)Endocrinology 103, 240-245; Markaverich B M and Clark J H (1979)Endocrinology 105, 1458-1462; Katzenellenbogen B S (1980) Annu RevPhysiol 42, 17-35; Katzenellenbogen B S (1984) J Steroid Biochem 20,1033-1037; Clark J H and Markaverich B M (1983) Pharm Ther 21, 429-453;Darbre P et al. (1983) Cancer Res 43, 349-355; Darbre P D et al. (1984)Cancer Res 44, 2790-2793; Huseby R A et al. (1984) Cancer Res 44,2654-2659; Gorski J and Hansen J C (1987) Steroids 49, 46.1-475;Katzenellenbogen B S et al. (1987) Cancer Res 47, 4355-4360; O'Malley BW (1990) Mol Endocrinol 4, 363-369). As discussed elsewhere herein, thepreferred positive action of estrogens is activation of a new ERγ thatsaturates/activates at lower steroid concentrations than the ERα or theERβ.

Serum Proteins with Estrocolyone Steroid Binding Characteristics.

If the estrocolyone mechanism is in fact correct, one must be able toidentify at least one serum protein with very high affinity binding(i.e. K_(d) picomolar) for sex steroids. There is, however, a majorunresolved problem with that hypothesis. Other than sex hormone bindingglobulin (SHBG), additional high affinity estrogen binding in CDE humanserum has not been found. SHBG has K_(d) of 1.7×10⁻⁹ M for E₂ at 37° C.(Rosner W and Smith R N (1975) Biochemistry 14, 4813-4820). Thisaffinity does not qualify as the high binding expected of estrocolyone.Also, a search for estrocolyone in human serum only resulted inidentification of SHBG (Reny J-C and Soto A M (1992) J Clin EndocrinolMetab 68, 938-945). No higher affinity binding site/protein was found.The binding of labeled steroid hormones with CDE-horse and CDE-rat serumwas studied (results presented in an Example which follows), and ³H-E₂specific binding at K_(d) of 20 to 50 nM was found. This is asignificant matter because estrogenic effects are demonstrated in thisdisclosure at 1 to 10 picomolar. As further support for this point, theestrocolyone authors found estrogenic effects at 10 to 30 picomolar E₂(Soto A M and Sonnenschein C (1985) J Steroid Biochem 23, 87-94; Soto AM and Sonnenschein C (1987) Endocr Rev 8, 44-52). The lack ofcorrelation between the concentration of steroid that promotes growthand affinity of sex steroids for serum components raises seriousquestions about this aspect of the estrocolyone hypothesis. Theseobservations also suggest that a very high affinity intracellular ERγregulates growth.

A New Model of Steroid Hormone Responsive Cell Growth.

A new model best fits the available data. It brings together aspects ofboth the direct positive mechanism and indirect negative control.According to this model, regulation of steroid hormone target tumor cellgrowth is a balance between positive and negative control signals. Thisbalance dictates either growth (i.e. cell division) or quiescence (i.e.cell metabolism and tissue specific function but without cell division).Direct positive control is mediated by a high sensitivity intracellularsex steroid receptor (yet to be defined) that ultimately activates geneexpression whereas negative regulation is exerted by a serum-borneinhibitor that acts at the cell surface. The results disclosed hereinsupport the view that growth is controlled directly by both negative andpositive mediators. The results presented further define the molecularproperties of the serum-borne inhibitor by eliminating TGFβ1 as acandidate. This is an important issue because of the well-known effectsof TGFβ on normal breast epithelial cells (Hosobuchi M and Stampfer M R(1989) In Vitro Cell Dev Biol 25, 705-713) and ER⁻ estrogen insensitivebreast cancer cells (Arteaga C L et al. (1988) Cancer Res 48,3898-3904). The results herein continue to confirm a previouslyunrecognized entity that serves as the estrogen reversible inhibitor inserum. Inhibitors that lack estrogen reversibility can be eliminatedfrom consideration.

Example 9 Serum-Free Defined Culture Medium Formulations

In this Example, formulations of various serum-free defined culturemedia are discussed. Among other features, the preferred embodiments ofthe present media provide useful tools for detecting estrogenic effects.

Serum-Free Defined Mammalian Cell Culture—Development Background.

The use of serum-free defined medium to grow diverse cell types inculture gained national and international recognition with thepublication by Hayashi and Sato (Hayashi I and Sato G H (1976) Nature(Lond) 259, 132-134). They demonstrated a breakthrough. The serumsupplement commonly used in cell culture medium could be replaceableentirely by mixtures of nutrients and hormones in serum-free medium.This observation was expanded to include cell types from many mammaliantissues (Barnes D and Sato G (1980) Anal Biochem 102, 255-270; Barnes Dand Sato G (1980) Cell 22, 649-655; Bottenstein J et al. (1979) MethodsEnzymol 58, 94-109; Rizzino A et al. (1979) Nutr Rev 37, 369-378).Further development and application of this technology has been reported(Barnes D W, Sirbasku D A and Sato G H (Volume Editors) (1984) CellCulture Methods for Molecular Biology and Cell Biology, Volume 1:Methods for Preparation of Media, Supplements, and Substrata forSerum-free Animal Cell Culture; Volume 2: Methods for Serum-free Cultureof Cells of the Endocrine System; Volume 3: Methods for Serum-freeCulture of Epithelial and Fibroblastic Cells; Volume 4: Methods forSerum-free Culture of Neuronal and Lymphoid Cells, Allan R. Liss/JohnWiley, New York). A national/international symposium organized anddirected by Drs. Gordon Sato, Arthur Pardee and David Sirbasku was heldat the Cold Spring Harbor Laboratory to address the unfolding technologyrequired for serum-free defined medium growth of cells in culture and todiscuss its applications (Sato G H, Pardee A B and Sirbasku D A (1982)Volume Editors, Cold Spring Harbor Conferences on Cell Proliferation,Volume 9, Books A and B, Growth of Cells in Hormonally Defined Media,Cold Spring Harbor, N.Y.).

Serum-Free Defined Culture—Nutrient Additions.

A number of nutrient additions to D-MEM/F-12 are needed to grow thecells used in the presently described studies. The formulations ofserum-free defined medium employed are specific optimizations,modifications, or necessary changes of earlier media that have beendescribed (Riss T L and Sirbasku D A (1987) Cancer Res 47, 3776-3782;Danielpour D et al. (1988) In Vitro Cell Dev Biol 24, 42-52; Ogasawara Mand Sirbasku D A (1988) In Vitro Cell Dev Biol 24, 911-920; Karey K Pand Sirbasku D A (1988) Cancer Res 48, 4083-4092; Riss T L et al. (1988)In Vitro Cell Dev Biol 24, 1099-1106; Riss T L et al. (1988) In VitroCell Dev Biol 25, 127-135; Riss T L and Sirbasku D A (1989) In VitroCell Dev Biol 25, 136-142; Riss T L et al. (1986) J Tissue CultureMethods 10, 133-150; Sirbasku D A et al. (1991) Mol Cell Endocrinol 77,C47-055; Sirbasku D A et al. (1991) Biochemistry 30, 295-304; Sirbasku DA et al. (1991) Biochemistry 30, 7466-7477; Sato H et al. (1991) InVitro Cell Dev Biol 27A, 599-602; Sirbasku D A et al. (1992) In VitroCell Dev Biol 28A, 67-71; Sato H et al. (1992) Mol Cell Endocrinol 83,239-251; Eby J E et al. (1992) Anal Biochem 203, 317-325; Eby J E et al.(1993) J Cell Physiol 156, 588-600; Sirbasku D A and Moreno-Cuevas J E(2000) In vitro Cell Dev Biol 36, 428-446).

Serum-Free Defined Medium Nutrient Supplements—Bovine Serum Albumin.

Bovine serum albumin (BSA) (Sigma Catalog No. A3912) was made by“initial fractionation by heat shock and Fraction V”, minimum purity 98%(electrophoresis), according to the supplier. A 50 mg/mL stock solutionof BSA was prepared in normal saline and was sterilized using 0.2 μmpore membrane filters. Aliquots are stored at −20° C. in plastic tubes.As will be discussed below, the “heat shock” step that was used in mostalbumin preparation methods inactivates the estrogen reversibleinhibitor disclosed herein.

Serum-Free Defined Medium Nutrient Supplements—Linoleic Acid—Albumin(Lin-Alb).

This preparation was purchased from Sigma as Linoleic Acid AlbuminConjugate (Catalog No. L8384). The conjugate is supplied as a powdersterilized by irradiation. The fatty acid content is 1% (w/w) linoleicacid. A stock solution was typically prepared by dissolving the contentsof a 500 mg bottle in 10 mL of sterile normal saline to give a finalconcentration of 50 mg/mL. Aliquots are stored at 4° C. in polystyrenetubes. This solution is never frozen. Mammalian cells cannot producepolyunsaturated fatty acids. They must be supplied in a soluble form.Fatty acids are carried physiologically bound to albumin.

Serum-Free Defined Medium Nutrient Supplements—Ethanolamine (ETN).

ETN was purchased from Sigma (Catalog No. A5629) (FW 61). This liquidhas a density of 1.0117 grams/mL. Using 0.610 mL in 100 mL of water, a100 mM stock solution was prepared which was sterilized using the 0.2 umpore membrane filters. The ETN was stored at −20° C. in polystyrenetubes. This nutrient is required to sustain phospholipid metabolismrequired for all membrane biosynthesis.

Serum-Free Defined Medium Nutrient Supplements—Phosphoethanolamine(PETN).

This solid material was purchased as o-phosphoryl-ethanolamine (FW 141)(Sigma Catalog No. P0503). A 10 mM stock of PETN was prepared bydissolving 141 mg in 100 mL of water and sterilizing with 0.2 μm poremembrane filters. Aliquots were stored at −20° C. in polystyrene tubes.This component is an adjunct to ETN.

Serum-Free Defined Medium Nutrient Supplements—Glutamine (GLUT).

This essential amino acid was purchased from Sigma (Catalog No. G5763).It is “cell culture tested” according to the manufacturer. Addition ofglutamine (FW 146.1) to the culture media is necessary because of itsrelatively short half-life (i.e. about 80% is lost in 20 days at 35°C.). See the Sigma product information for the decay curves at differenttemperatures and pH. Purchased D-MEM/F-12 stored in the refrigerator forabout three weeks lost most of the original glutamine present. Forserum-free applications, additional supplementation is required tosustain growth. For a preparation, 11.7 g was dissolved in 400 mL ofwater to give 200 mM glutamine. This solution was sterilized using 0.2μm pore filter membranes. Aliquots are stored at −20° C. polystyrenetubes. The final glutamine concentration added to serum-free definedmedium is 2 mM. Glutamine is a major metabolite and energy source forcells growing in culture.

Serum-Free Defined Medium Nutrient Supplements—Reduced Glutathione(GSH).

Crystalline reduced glutathione (FW 307.3) was purchased from Sigma(Catalog No. G4251). A stock of 40 mg/mL was prepared by dissolving 400mg in 10 mL of water. This stock was very quickly sterilized with a 0.2μm pore filter unit. Aliquots were quickly stored at −20° C. inpolystyrene tubes. According to Sigma technical service, this sulfhydryl(—SH) compound is unstable in aqueous solutions, including tissueculture medium, and is rapidly converted to the oxidized GS-SG form byexposure to air. Addition every two to four days to the culture mediummay be required for reducing agent requiring cells. Another reducingagent that also is effective is mercaptoethanol. It is more stable andoften effective at lower concentrations than GSH. Preferably theconcentrations are controlled effectively. Reducing agents act as“scavengers” of free radicals generated by the oxygen atmosphere of cellculture.

Serum-Free Defined Medium Nutrient Supplements—Selenium (Se).

A powder of sodium selenite (100 mg/vial) is obtained from CollaborativeResearch or Sigma (Catalog No. S5261). It has been sterilized byirradiation. The contents of a single vial are dissolved in 100 mL ofsterile water to give final stock of 1.0 mg/mL. This preparation shouldnot be filter sterilized because Se binds to filters. The final volumewas diluted to 100 mL with sterile saline. Aliquots are stored at −20°C. in polystyrene tubes. Selenium is an important cofactor for enzymesystems that protect the cells from oxidation effects.

Serum-Free Defined Medium Nutrient Supplements—Diferric Transferrin(2FeTf).

Iron Fe (III) saturated (98%) human transferrin (diferric transferrin)was purchased from Collaborative Research (Catalog No. 40304) or Sigma(Catalog No. T3309) as bottles containing 1 gram of red colored powder.The contents of one bottle are dissolved in 100 mL of normal saline.This red colored solution is sterilized using 0.2 μM pore membranefilters. This stock is 10 mg/mL. Aliquots are stored at −20° C. inpolystyrene tubes. All growing cells require diferric transferrin as asource of iron for a great many metabolic processes, except for a fewknown cell types in which free Fe (III) or chelated Fe (III) can besubstituted for diferric transferrin. The cell lines employed in thepresent Examples do not include those exceptional cell types, however.

Serum-Free Defined Medium Growth Factor Supplements—Epidermal GrowthFactor (EGF).

EGF prepared from mouse submaxillary gland (tissue culture grade) waspurchased from Collaborative Research (Catalog No. 40001) as 100 μg in asterile vial or from Sigma (Catalog No. E4127). The original vials arestored at 4° C. according to the manufacturer's instructions. To preparea stock solution, 5.0 mL of sterile saline was added to a vial to yielda 20 μg/mL EGF solution. Aliquots are stored frozen at −20° C.polystyrene tubes. Repeated freeze-thaw must be avoided. This growthfactor is useful because of its very broad cell specificity range.

Serum-Free Defined Medium Growth Factor Supplements—Acidic FibroblastGrowth Factor (aFGF).

Acidic FGF is purchased from Sigma (Catalog No. F5542). It is the humanrecombinant product from E. coli. This product has very specifichandling requirements. It is provided sterilized in 25 μg vialslyophilized from PBS containing 1.25 mg of BSA. The contents of eachvial are reconstituted in 25 mL of sterile PBS containing 1.0 mg/mL ofBSA and 10 μg/mL of heparin. Filtration of this product at thisconcentration must absolutely be avoided. This solution is stored at−20° C. in polystyrene tubes. The solutions of aFGF definitely cannot befreeze-thawed more than twice. This growth factor is highly labile.Careless handling will result in problems. Keratinocyte growth factor(KGF) can substitute for aFGF. The fibroblast growth factor family isimportant in growth of urogenitial tissues including bladder andprostate (Liu W et al. (2000) In Vitro Cell Dev Biol 36, 476-484).

Serum-Free Defined Medium Growth Factor Supplements—Heparin.

Heparin is used to stabilize FGF in cell culture (Gospodarowitz D andCheng J (1986) J Cell Physiol 128, 475-484). Heparin is obtained fromSigma (Catalog No. H3149) as the sodium salt, Grade 1-A, from porcineintestinal mucosa. A solution of 1.0 mg/mL is made in saline andsterilized with 0.2 μm pore membrane filters. An aliquot of 250 μL isadded to the 25 mL of aFGF reconstitution solution used above. Sterileheparin is stored at 4° C.

Serum-Free Defined Medium Adhesion Protein Supplement—Fibronectin (Fbn).

Human plasma derived fibronectin can be purchased from many commercialsources. Bovine fibronectin is also available and is effective.Fibronectin is prepared from units of fresh human plasma (unfrozen) orfresh bovine (unfrozen) plasma by two methods (Retta S F et al. (1999)Methods in Molecular Biology 96, 119-124; Smith R L and Griffin C A(1985) Thrombosis Res 37, 91-101). Purity is evaluated by SDS-PAGE withCoomassie Brilliant Blue staining or silver staining (Pierce Chemicals®kits). Adhesion activity is confirmed with cells in serum-free definedmedium. Vitronectin can substitute for fibronectin.

Serum-Free Defined Medium Iron (Fe (III) ChelatorSupplements—Deferoxamine Mesylate (DFX).

Deferoxamine (FW 656.8) is purchased from Sigma (Catalog No. D9533). Thestock solution is made at 10 mM by adding 131 mg to 20 mL of highlypurified water as described above. The solution is sterilized byfiltration with 0.2 μM pore membranes. Aliquots are stored at −20° C. inpolystyrene tubes.

Serum-Free Defined Medium Iron (Fe (III) ChelatorSupplements—Apotransferrin (apoTf).

Human serum apotransferrin can be purchased from Sigma (Catalog NoT4382). It is minimum 98% iron-free. Alternatively, apotransferrin isprepared, as described previously (Sirbasku D A et al. (1991)Biochemistry 30, 295-304; Sirbasku D A et al. (1991) Biochemistry 30,7466-7477). Apotransferrin is prepared by dialysis against citratebuffer pH 5.0-5.5 with 1 μg/mL DFX present to chelate >98% of the iron.Handling and storage were as described for diferric transferrin but withgreat care to avoid contact with iron sources.

Serum-Free Defined Medium Nutrient Supplements—Bovine Insulin (INS).

This hormone was purchased from either of two sources. From Gibco-BRL itis Insulin, Bovine Zinc Crystals for Cell Culture Applications (CatalogNo. 18125-039). It was also obtained from Collaborative Research(Catalog No. 40305) and stored at 4° C., according to thatmanufacturer's recommendation. Gibco-BRL recommends solid insulinstorage at −5° C. to 20° C. A stock of 10 mg/mL in 0.01 N HCl wasprepared by adding 250 mg of insulin to 25 mL of the acid. The HCl wasmade by adding 172 μL of concentrated (11.6 N) HCl to 100 mL of water.The final stock solution of 10 mg/mL of insulin is filter sterilizedusing 0.2 μm pore diameter membranes. Aliquots are stored at 4° C. inpolystyrene tubes. Care was taken not to freeze-thaw the aliquots ofstock solution. Insulin is a very broad range cell growth-stimulatingfactor as well as a regulator of specific metabolic processes. Atsufficiently high concentrations (i.e., usually >1 μg/mL, insulin causesgrowth via binding to the IGF-I Type I receptor (Karey K P and SirbaskuD A (1988) Cancer Res 48, 4083-4092).

Serum-Free Defined Medium Nutrient Supplements—Thyroid Hormones.

The preferred thyroid hormone is T₃ (3′,5-Triiodothyronine (FW 673)),purchased from Sigma as Catalog No. T2752). It is stored desiccated at−20° C. To prepare stocks, 0.5 N NaOH was made by addition of 20 gramsof pellets to one liter of water. Then, 67.3 mg of T₃ was added. Afterdissolving the T₃ with stirring for a few minutes, 25 mL of this stockwas diluted up to 250 mL with water, for a final concentration of 0.05 NNaOH. This dilution was sterilized using the 0.2 μm pore diameterfilter. At this point, the final stock for storage was 10 μM T₃.Aliquots of this final stock are stored in polystyrene tubes at −20° C.The second thyroid hormone, thyroxin (T₄, sodium salt, pentahydrate FW888.9), is prepared by the same procedure. For this stock solution, 88.9mg of T₄ are used. T₄ is purchased from Sigma (Catalog No. T2501). T₄ isused at 10 to 20 times higher concentrations than T₃. Care is taken notto freeze-thaw these preparations. Thyroid hormones have a very broadrange of metabolic and growth effects, and many different types of cellsrequire thyroid hormones for growth in serum free culture.

Compositions of Serum-Free Defined Media.

TABLE 6 presents the formulations of the preferred serum-free definedmedia developed for use in detecting high-level steroid hormonereversible inhibition by steroid hormone-depleted (“steroid hormonestripped”) serum fractions and by purified inhibitors in serum-free cellgrowth assays. As indicated in the footnotes to the table, when aparticular component is included in one of the formulations, theconcentration that provides a suitable cell growth medium can fallwithin the indicated range.

TABLE 6 Composition of Serum-free Defined Media Based on StandardGibco-BRL D-MEM/F-12 CELL TYPE Human Human Rat Rat Hamster BreastProstate Mammary Pituitary Kidney MEDIUM NAME DDM-2MF CAPM DDM-2A PCM-9CAPM COMPONENT FINAL CONCENTRATIONS IN THE DEFINED MEDIA Insulin¹ 500ng/mL 10 μg/mL 10 μg/mL 10 μg/mL 10 μg/mL EGF² 20 ng/mL 20 ng/mL 20ng/mL None 20 ng/mL AFGF³ None 10 ng/mL None None 10 ng/mLTriiodothyronine⁴ 0.3 nM 1.0 nM 0.3 nM 1.0 nM 1.0 nM Diferrictransferrin⁵ 10 μg/mL 10 μg/mL 10 μg/mL 10 μg/mL 10 μg/mL Ethanolamine⁶50 μM 50 μM 50 μM 10 μM 50 μM Phosphoethanolamine⁷ 5 μM None 5 μM NoneNone Bovine Serum Albumin⁸ 500 μg/mL 1.0 mg/mL 500 μg/mL 500 μg/mL 1.0mg/mL Linoleic acid-BSA⁹ 150 μg/mL None 150 μg/mL None None Selenium¹⁰20 ng/mL 10 ng/mL 20 ng/mL 10 ng/mL 10 ng/mL Reduced glutathione¹¹ 20μg/mL None 20 μg/mL None None Glutamine¹² 2.0 mM None 2.0 mM None NoneHeparin¹³ None 7.5 μg/mL None None 7.5 μg/mL Deferoxamine¹⁴ 5 μM 10 μM 5μM 10 μM 10 μM Human Fibronectin¹⁵ 25 μg 20 μg None None 20 μg When acomponent is added, the following are the effective concentration rangesused: ¹INS range 100 ng/mL to 10 μg/mL ²EGF range 1 ng/mL to 50 ng/mL³aFGF range 0.2 ng/mL to 20 ng/mL ⁴T₃ range 0.3 nM to 10 nM ⁵2FeTf range2 μg/mL to 50 μg/mL ⁶ETN range 5 μM to 100 μM ⁷PETN range 5 μM to 50 μM⁸BSA range 0.2 mg/mL to 5.0 mg/mL ⁹Lin-Alb range 50 μg/mL to 500 μg/mL¹⁰Se range 5 ng/mL to 20 ng/mL ¹¹GSH range 1 μg/mL to 50 μg/mL ¹²Glutrange 0.5 mM to 2.0 mM ¹³Heparin range 1 μg/mL to 10 μg/mL ¹⁴DFX range 2μM to 20 μM ¹⁵Fbn range 15 μg to 50 μg per 35-mm diameter dish

Serum-Free Media Variations.

The variations described next are applicable to the defined media inTABLE 6. Standard phenol red-containing Gibco-BRL D-MEM/F-12 is apreferred basal medium to which the defined media components are added.It contains 0.6 mM to 1.0 M CaCl₂. D-MEM/F-12 can be purchased fromGibco-BRL in the liquid form or can be prepared from the powderformulation using only highly purified water. Alternatively, anothersuitable basal medium could be used as long as it provides at least therequired minimum amounts of necessary nutrients, vitamins and mineralsto maintain cell viability of the desired cell line. The calciumconcentration range preferred is 0.6 to 10 mM. Calcium stabilizes theinhibitor in cell culture without impairing cell growth. The humanbreast cancer cell medium, DDM-2MF, was a modification of the originalDDM-2 medium (Danielpour D et al. (1988) In Vitro Cell Dev Biol 24,42-52) and MOM-1 (Ogasawara M and Sirbasku D A (1988) In Vitro Cell DevBiol 24, 911-920) and contained modified hormone concentrations,deferoxamine (DFX) and fibronectin. Aqueous salt solutions such astissue culture medium contain hydrolytic polymeric forms of Fe (III)(Spiro T G et al. (1966) J Am Chem Soc 88, 2721-2726). DFX binds thisform of Fe (III) with very high affinity (Schubert J (1964) In; IronMetabolism The Chemical Basis of Chelation, Springer, Berlin, pp466-498). If not removed, Fe (III) inhibits hormone-responsive growth inserum-free defined medium (Sirbasku D A et al. (1991) Mol CellEndocrinol 77, C47-C55; Sato H et al. (1992) Mol Cell Endocrinol 83,239-251; Eby J E et al. (1993) J Cell Physiol 156, 588-600; Eby J E etal. (1992) Anal Biochem 203, 317-325). The preferred cell growth mediafor conducting cell growth assays are substantially devoid of unbound Fe(III), i.e., preferably containing less than 1 μM Fe (III), and morepreferably containing no more than about 0.15 μM. In preferred growthassay systems described herein, which are substantially devoid ofunbound Fe (III), the concentration of free, or active Fe (III) in themedium is less than a cell growth inhibiting amount. Fibronectin wasused with DDM-2MF to promote cell attachment. The 35-mm diameter assaydishes were pre-coated by incubation with the designated amount offibronectin (TABLE 6) for 16 to 48 hours at 37° C. in 2.0 mL ofD-MEM/F-12. CAPM human prostatic cancer cell medium was developed tosupport the growth of tumor cells from this tissue. The composition ofCAPM is described in TABLE 6. CAPM also supports the growth of the H301Syrian hamster kidney tumor cells. DDM-2A, which is a modified form ofDDM-2 (Danielpour D et al. (1988) In Vitro Cell Dev Biol 24, 42-52), waspreferred for growing MTW9/PL2 cells. PCM-9 defined medium was developedfor growing the rat pituitary cell lines. This medium differs fromprevious PCM formulations (Sirbasku D A et al. (1991) Mol CellEndocrinol 77, C47-055; Sato H et al. (1992) Mol Cell Endocrinol 83,239-251; Eby J E et al. (1993) J Cell Physiol 156, 588-600; Eby J E etal. (1992) Anal Biochem 203, 317-325) in that DFX was substituted forapotransferrin and the triiodothyronine concentration was increased to1.0 nM. Although DFX and apotransferrin (2 to 50 μg/mL) are thepreferred chelators based on their very high specificity and affinitiesfor Fe (III), EDTA at 1 to 10 μM or sodium citrate at 10 to 1000 μM alsoeffectively neutralize the cytotoxic effects of Fe (III) (Eby J E et al.(1993) J Cell Physiol 156, 588-600). Ascorbic acid (vitamin C) alsochelates Fe (III), but is used less often because it is unstable in cellculture medium at 37° C. in an oxygen environment in the presence ofsalts and metals in the medium. Also, at concentrations of 50 to 100μg/mL, apo-ovotransferrin and apo-lactoferrin also were effective Fe(III) chelators in serum-free defined medium (Eby J E et al. (1993) JCell Physiol 156, 588-600). Although EGF, aFGF and insulin are thepreferred growth factors, several other human recombinant proteins areeffective. They have either been purchased or obtained as gifts fromGibco-BRL, Sigma or IMCERA Bioproducts. Insulin-like growth factors Iand II (IGF-I and IGF-II) can be used to replace insulin, transforminggrowth factor α (TGFα) replaces EGF, TGFβ as an inhibitory supplement,and basic fibroblast growth factor (bFGF) partially replaces aFGF.Insulin can be used to replaced IGF-I and IGF-II. All of these proteingrowth factors are dissolved under sterile conditions according tomanufacturers' instructions and stored as indicated.

Discussion of Example 9

The preferred serum-free media described above provide an ideal scenariofor the study of growth responses of hormone responsive cancers withoutthe myriad of potential interactions accompanying the presence of serumwith its 5000+ proteins and other compounds. The formulations presentedpermit dissection of growth into its individual parts caused bydifferent stimulators. When of interest, a combination of a few factorscan be investigated to achieve an understanding of growthpromoter/inhibitor interactions (i.e. cross-talk). This is exceptionallydifficult to achieve in the presence of full serum. The serum-freemedium described herein provided a tool for the assessment of growthinhibitor(s) isolated from CDE-horse serum, whose actions are reversedby sex-steroid hormones, as mentioned at the beginning of this Exampleand also discussed elsewhere herein. These serum-free defined media willallow direct analysis of the final purified serum-borne inhibitors underthe most defined conditions available for cell culture. This featurebrings the regulation of steroid hormone dependence up to the conditionsthat have been the most sought after over the past fifteen years. Thepreferred serum-free media of the present invention raise hope for theprovision of new insight that could help to clarify the mechanismsinvolved in the control of breast, prostatic and other mucosal cancersunder conditions not previously available.

Moreover, because of widespread concern today about possiblecontamination of commercial animal sera by disease causing agents suchas bovine spongiform encephalopathy (“mad cow disease”), there is agreat need for serum-free cell culture media that can support a varietyof cell types. The new media compositions fill that need. The newserum-free media can be used not only for assays but also for largescale testing purposes and industrial uses such as cell cultureproduction of a desirable protein. For example, an antigen for vaccineproduction, or a monoclonal antibody can be prepared without fear ofcontamination by a serum-derived agent. The serum-free media are alsouseful for producing quantities of virus for vaccine manufacture or forproducing recombinant viruses for gene therapy, and can be substitutedfor a conventional serum-based medium in a basic cell culture method forproducing quantities of proteins or viruses. Such basic cell culturemethods are well known in the art and have been described in theliterature.

Example 10 Serum-Free Defined Medium Supports Both Hormone Sensitive andAutonomous Cancer Cell Growth

In this Example, it is shown that media derived according to the presentmethods are effective for supporting hormone sensitive and autonomouscancer cell growth.

Selection of Models to Study Hormone Dependence and Autonomy inSerum-Free Defined Culture Media.

One goal was to develop serum-free defined media that can be used todirectly compare negative serum factor regulation with steroid hormoneresponsive and steroid hormone autonomous cancers of the same tissue.That meant establishing a medium that supported the growth of both celltypes. As models, human prostatic carcinoma and human breast carcinomacells were chosen because responsive and autonomous (unresponsive) celllines are currently available for both types of cancers. Furthermore, asdiscussed above, these cancers have many common characteristicsincluding their tendency to pass from steroid hormone receptor positiveto steroid hormone receptor negative in a process called tumorprogression. During the course of development of such defined media, oneobservation was made consistently: breast cancer cells that were ER⁺(i.e. estrogen sensitive) and prostate cancer cells that were AR⁺ (i.e.androgen sensitive) grew less well in defined medium based on standardD-MEM/F12 than in defined medium based on “low-Fe” D-MEM/F12. Theresults of an example with T47D cells in DDM-2MF are shown in FIG. 29.The example with LNCaP cells in CAPM is shown in FIG. 30. Anotherexample is the thyroid hormone responsive MDCK kidney tubule epithelialcells in CAPM as shown in FIG. 31. Standard D-MEM/F-12 contains bothferric nitrate and ferrous sulfate as nutrient additions. When purchasedwithout these salts, the medium was designated “low-Fe” D-MEM/F-12. Theiron concentrations in standard and “low-Fe” D-MEM/F-12 were 1.0 μM and0.15 μM, respectively (Eby J E et al (1992) Anal Biochem 203, 317-325).Even in “low-Fe” medium, iron is present as a contaminant in thechemicals used to make the formulation, the 2.2 g/L NaHCO₃ added as ametabolic requirement and buffer, and the 15 mM HEPES buffer necessaryfor stabilizing the pH under serum-free conditions (Eby J E et al (1992)Anal Biochem 203, 317-325). It is noteworthy that as low as 1.0 μM Fe(III) inhibits epithelial cell growth completely within five to sevendays. In another test the thyroid hormone responsive human HT-29 coloniccarcinoma cells in CAPM also grew better in “low-Fe” than standardD-MEM/F-12 (data not shown). This indicates that restriction of Fe (III)in culture medium will have implications even beyond sex steroid hormonedependent cells.

Modifications of the Usual Growth Assays for Experiments in “Low-Fe”Medium Versus “Standard” Medium.

Specific modifications of the customary cell growth assays were requiredfor assays done under iron-restricted conditions. For example, the 35-mmassay dishes were incubated for 16 to 24 hours prior with 20 to 25 μg offibronectin in 2 mL of “low-Fe” D-MEM-F12 medium. Serum-free componentswere added to “low-Fe” D-MEM/F-12 at double the concentrations needed(2×) or to “standard” D-MEM/F-12 at (2×) as the experiments dictated.Each assay dish received 1.0 mL of this solution. Next, the cells to beused in the assays were washed three times in either “low-Fe” medium or“standard” medium depending upon the experimental protocol. These washeswere done with the same care as discussed above in the general materialsand methods described in Example 1. Each dish received 1.0 mL of cellsin the appropriate medium. At this point, the components finalconcentrations were (1×) as summarized in TABLE 6. Also, TABLE 6describes medium containing deferoxamine as the Fe (III) chelator.Although less preferred, due in part to cost considerations,specificity, and affinity for Fe (III), as noted above, apotransferrinis also effective, especially at the preferred apotransferrinconcentration of 50 μg/mL. When apotransferrin binds Fe (III), it isconverted to one of three forms of ferric transferrin (Eby J E et al(1992) Anal Biochem 203, 317-325). These three forms become additionalsupport for cell growth in defined medium, thereby converting a toxicsubstance to a useable natural nutrient.

Growth in Serum-Free Defined Medium Versus D-MEM/F-12 with 10% (v/v)Fetal Bovine Serum.

To demonstrate the utility of the formulations in TABLE 6, cell growthwas compared in serum-free defined medium±steroid hormone versus growthsupported by fetal bovine serum. It is generally accepted that fetalbovine serum represents one of the most effective sera for tissueculture. As an example, growth of the LNCaP cells was compared inCAPM±DHT versus growth in 10% (v/v) fetal bovine serum (FIG. 32). CAPMplus 10 nM DHT supported growth at about 80 to 90% of the rate of fetalbovine serum. Growth promoted by 10% fetal bovine serum, typicallyobtained from conventional commercial sources, reached 6.57 (±0.48) CPDor, a 96-fold increase on cell number in 12 days. By day 12, celldensities in CAPM nearly equaled those in serum. Growth promoted by theserum-free medium reached 6.22 (±0.35) CPD or 84-fold increase. CAPM wasable to support LNCaP growth even in the absence of sex-steroidhormones. Maximum growth obtained without sex-steroid hormones was of5.35 (±0.12) CPD or a 49-fold increase. The androgenic effect istherefore marginal, with differences of less than one CPD between thepresence and absence of DHT. Also shown, the cells did not grow inD-MEM/F-12 without any additions (FIG. 32). Similar studies were donewith other cell lines to determine growth rates versus serum and toestablish the periods for single time assays (e.g. 7, 10, 12 or 14days). FIG. 33 shows the same analysis with DU145 and PC3 cells in CAPMand in D-MEM/F-12 with 10% fetal bovine serum. As the cell number datashow, growth was logarithmic. After 12 days, growth in the serum-freemedium was identical to that in 10% fetal bovine serum for both celllines. Growth of PC3 in 10% serum reached 6.98 (±0.71) CPD or a 112-foldincrease in cell number versus 6.97 (±0.44) CPD or the same foldincrease for cell numbers in serum-free medium. Growth of DU145 in 10%fetal bovine serum was 6.71 (±0.58) CPD versus 6.73 (±0.18) CPD inserum-free conditions. The results in FIGS. 32 and 33 demonstrate byexample that the serum-free defined media in TABLE 6 are effective withboth hormone sensitive and hormone autonomous cells.

Determination of Component Concentrations and the Requirement for a Fe(III) Chelator.

The optimum concentration of each single component was determined bydose-response analysis in the presence of other components. Thetechnology used to establish early forms of serum-free defined media hasbeen described (Danielpour D et al. (1988) In Vitro Cell Dev Biol 24,42-52; Ogasawara M and Sirbasku D A (1988) In Vitro Cell Dev Biol 24,911-920). An example of this process is shown in FIG. 34 with LNCaPcells. Dose-response effects of bovine serum albumin, apotransferrin,T₃, ethanolamine, selenium, and EGF are shown. The results show clearlythat the addition of the iron chelator apotransferrin was required forcell growth. After determining optimum concentrations for eachcomponent, the contribution of each to the total was assessed by anotherassay. Individual components were deleted one at a time. As an example,the three most widely used prostatic carcinoma cell lines were compared(i.e. LNCaP, PC3 and DU145) in CAPM that contained deferoxamine in placeof apotransferrin (FIG. 35). The deletions were done±DHT. The first andmost striking result was the major differences between the growthrequirements of the DHT sensitive LNCaP cells and those of theautonomous DU145 and PC3. Only the deletion of diferric transferrinsubstantially prevented the growth of autonomous cells. Also, it wasclear that deletion of deferoxamine had only a small (i.e. <20%) effecton growth of the DU145 and PC3 cells. The DU145 and PC3 cell lines alsowere T₃, insulin, EGF, fibronectin and deferoxamine independent. Asexpected±DHT had no significant effect on DU145 or PC3. By contrast,LNCaP growth was significantly (p<0.01) reduced or arrested completelyby deletion of fibronectin, T3, diferric transferrin or deferoxamine.LNCaP growth also was inhibited by deletion of EGF or insulin, but theseeffects were pronounced only in the absence of DHT.

Discussion of Example 10

The media described in TABLE 6 were optimized for the specific celltypes designated. Additionally, they were optimized to permit directcomparison of the growth properties of ER⁺ and AR⁺ steroid hormonesensitive tumor cell lines to their ER⁻ and AR⁻ steroid hormoneinsensitive (also called autonomous) counterparts. This carefuloptimization was done originally to study rat mammary tumor cells ofboth types in DDM-2A defined media. The appropriate cell lines for thisapproach have been developed from the MTW9/PL2 population and described(Danielpour D and Sirbasku D A (1984) In Vitro 20, 975-980). The mediumDDM-2MF has been developed for the same purpose only for comparisons ofER⁺ and ER⁻ forms of these cancers. TABLE 1 lists the most important ER⁺human breast cancer cell lines in use today. In addition a number ofother ER⁻ human breast cancer cells lines have been evaluated. They arethe MDA-MB-231 (Cailleau R et al. (1974) J Natl Cancer Inst 53,661-674), BT-20 (Lasfargues E Y and Ozzello L (1958) J Natl Cancer Inst21, 1131-1147), Hs0578T (Hackett A J et al. (1977) J Natl Cancer Inst58, 1795-1806), MDA-MD-330 (Cailleau R et al. (1978) In Vitro 14,911-915), and the myoepithelial HBL-100 (Gaffney E V (1982) Cell TissueRes 227, 563-568). The demonstration of ER status of these lines hasbeen described (Reddel R R et al. (1985) Cancer Res 45, 1525-1531). Withregard to human prostatic cancer, the only reliable androgen responsivecell line available today is the LNCaP (TABLE 1). Another, the ALVA-41,has been described as androgen growth responsive (Nakhla A M and RosnerW (1994) Steroids 59, 586-589). However, as shown in subsequentExamples, this line is autonomous by the criterion of a lack of DHTeffects in CDE-horse serum. Two other human prostate cancer cell linesare commonly used as autonomous examples. These lines are the DU145(Stone K R et al. (1978) Int J Cancer 21, 274-281) and the PC3 (Kaighn ME et al. (1979) Invest Urol 17, 16-23). Previously, there was a definedmedium established for PC3 cells (Kaighn M E et al. (1981) Proc NatlAcad Sci USA 78, 5673-5676). This medium was evaluated and did notsupport LNCaP cell growth. However, others have reported “serum-free”media that was stated to be effective with LNCaP, DU145, PC3 and ALVA-31cells (Hedlund T E and Miller G J (1994) The Prostate 24, 221-228). Theproblem was this medium was not serum-free nor was it defined. Theexperiments began with cells plated into 5% serum and then preceded touse a serum fraction called fetuin to support growth. Fetuin is acomplex undefined mixture of ≧4% of the proteins in serum. Under thoseconditions, an accurate analysis of hormonal and growth factor effects(Ogasawara M and Sirbasku D A (1988) In Vitro Cell Dev Biol 24, 911-920)cannot be done satisfactorily. The completely serum-free CAPM in TABLE 6supports the growth of all of these prostate cell lines. In addition,CAPM has been applied to the ER⁺ estrogen growth stimulated H301 Syrianhamster kidney cells (Sirbasku D A and Moreno J E (2000) In Vitro CellDev Biol 36, 428-446) and its autonomous derivative cell line A195. Ashas been reviewed (Evans R M (1988) Science (Wash DC) 240, 889-895),steroid hormones and thyroid hormones belong to the same superfamily ofreceptors. Both are important in growth. Therefore, it was expected thatsome tissues might be thyroid hormone positive regulated, while othersmight be positive regulated by steroid hormones. CAPM has also beenapplied to the study of thyroid hormone reversal of purified inhibitorswith the human colon carcinoma cell line HT-29. Similar use has beenmade of CAPM with the MDCK dog kidney tubule cell line (Leighton J etal. Science (Wash DC) 158, 472-473). CAPM replaces a different definedmedium prepared for MDCK cells (Taub M et al. (1979) Proc Natl Acad SciUSA 76, 3338-3342). It is likely that the prostaglandin in that earliermedium interfered with the action of the thyroid hormones. In any case,that medium was not useful for demonstration of thyroid hormone reversalof purified MDCK cell growth inhibitors. All of these observationssupport the view that a series of uniquely optimized media have beenformulated to define the growth requirements of epithelial cells fromseveral of the very prominent cancers of humans. Furthermore, thetechnology developed promises application to the optimization of growthof other types cells from a variety of epithelial/mucosal tissues.Epithelial/mucosal cancers comprise 80% of those in humans.

Example 11 Differential Effects of Fe (III) on the Growth of HormoneResponsive and Autonomous Human Breast and Human Prostate Cancer Cells

This Example demonstrates that iron has an inhibiting effect on steroidresponsive cell growth, independent of the above-describedimmunoglobulin effects, and which is distinguishable from its effect onautonomous cells.

Approaches to Demonstration of Iron Toxicity.

Standard D-MEM/F-12 appeared to contain sufficient Fe (III) to inhibithormone responsive cell growth (FIGS. 29 and 30). Accordingly, otherapproaches were used to further demonstrate the deleterious effects ofFe (III) on hormone responsive tumor cell growth. To add Fe (III) toculture medium, it must be in a soluble form. Ferric ammonium citratewas selected for use. However, ferric ammonium sulfate is alsoeffective. Other salts such as ferric chloride or ferric nitrate orferrous sulfate can be used. Ferric ammonium citrate is a mixture thatcontains 16.6% of ferric iron by weight. The amount of mixture added toeach dish was adjusted to achieve the desired Fe (III) concentrations.Due to the light sensitivity of the mixture, the solutions were preparedfresh daily and the experiments carried out under restricted lightconditions. Also, the mixture was prepared in water. Buffers withoutphosphate may be used, but they are generally less effective due toformation of insoluble materials. The ferric mixtures and the ironchelators EDTA, deferoxamine mesylate and sodium citrate were purchasedfrom Sigma.

Iron Toxicity with Human ER⁺ Breast Cancer Cells.

In the first experiments, two ER⁺ cell lines were evaluated for Fe (III)sensitivity in DDM-2MF defined medium prepared with 10 μg/mLapotransferrin in place of the deferoxamine shown in TABLE 6. The effectof addition of ferric ammonium citrate on MCF-7A growth±E₂ at 10 days isshown in FIG. 36. Either with or without steroid hormone, Fe (III) wascompletely inhibitory at 10 μM. There were no viable cells in the dishesat ≧10 μM. The EI₅₀ of Fe (III) with MCF-7A cells was 5 to 7 μM. Asimilar analysis with T47D cells in DDM-2MF with 10 μg/mL apotransferrininstead of deferoxamine showed complete inhibition at 10 days with 2 μMFe (III) (FIG. 37). At ≧2 μM there were no viable cells in the disheseither with or without (±) E₂. The EI₅₀ of Fe (III) with T47D cells was1 μM.

Iron Toxicity with AR⁺ and AR⁻ Human Prostate Cancer Cell Lines.

The effect of Fe (III) on AR⁺ LNCaP cell growth was assessed in CAPMdefined medium in which apotransferrin. (500 nM) was substituted fordeferoxamine, and the results are shown in FIG. 38. Clearly, 10 μM Fe(III) arrested growth to seed density levels (i.e. 12,000 cells perdish) in a 12-day assay. The EI₅₀ for LNCaP cells was 5 μM. In anotherexperiment in CAPM, the effects of ferric ammonium citrate wereevaluated with AR⁺ LNCaP cells and AR⁻ PC3 and DU145 cells (FIG. 39).Again, Fe (III) inhibited LNCaP cells to seed densities levels by 8 to10 μM. However, effects on the androgen autonomous PC3 and DU145 cellswere markedly less (FIG. 39). Reductions of 10 to 30% in cell'number forPC3 and DU145, respectively, were observed in 10 μM Fe (III). Theinhibitory effects of Fe (III) on the androgen independent PC3, DU145and ALVA-41 cells were variable, and never as marked as with the steroidhormone responsive LNCaP cells. The insert in FIG. 39 shows acorrelation between hormone responsiveness and Fe (III) effects. Theresults show a correlation between iron effects and thyroid hormoneresponsiveness. LNCaP cells are T₃ responsive whereas PC3 and DU145 arenot.

Reversal of Fe (III) Inhibition by Iron Chelators.

The inhibitory/cytotoxic effects of Fe (III) were reversible by theaddition of iron chelators. Those studied were selected based on datashowing their relative affinities and specificities for Fe (III)(Schubert J (1963) In: Iron Metabolism, Gross F, ed, Springer-Verlag,Berlin, pp 466-496). Deferoxamine is most specific and has the highestaffinity for Fe (III). Citrate is next most effective. EDTA is not aseffective nor is it as specific as the first two chelators. Inexperiments with T47D cells, the deferoxamine usually present in theDDM-2MF medium was removed and an additional 1.5 μM Fe (III) added toensure complete inhibition of the cells. FIG. 40 shows the relativeeffects of addition of these three chelators to T47D serum-free definedmedium cultures. The order of effectiveness was as expected from theaffinities and specificities of these chelators. Clearly, addition of Fe(III) chelators restored growth. FIG. 41 shows a similar study withLNCaP cells in CAPM defined medium from which the deferoxamine also wasremoved and 1.5 μM Fe (III) added. It was clear that chelation of the Fe(III) restored growth. It should be noted that this conclusion isreasonable based on the fact that deferoxamine has near absolutespecificity for Fe (III). Concentrations as low as 0.5 μM ofdeferoxamine were sufficient to induce 3.5 CPD with LNCaP cells. Maximumgrowth with this chelator (5.81 CPD) was obtained at 10 μM. Citrate andEDTA were also effective growth stimulators of LNCaP cells incubated athigh iron concentrations. Their maximum effects were with the additionof 500 μM and 10 μM respectively. The growth induction achieved withEDTA is lower than with citrate or deferoxamine. This probably could beexplained by the fact that EDTA is a less discriminatory chelator, andessential metals other than iron were affected. Concentrations of thechelators higher than those shown in FIGS. 40 and 41 were associatedwith cell damage and death. In particular, chelation of calcium bycitrate and EDTA will cause cell death in culture. The effect of thechelators was prevented by addition of more Fe (III) (data not shown).

Correlation Between Hormone Autonomy and Lack of Iron Effects.

In the next series of studies, data was sought supporting the conceptthat loss of steroid hormone dependence correlates positively with lossof Fe (III) effects. As shown in FIG. 30, LNCaP cells grew better in“low-Fe” serum-free defined medium than in defined medium based on“standard” D-MEM/F-12. This difference was also evaluated with theandrogen insensitive DU145 (FIG. 42) and PC3 (FIG. 43) cells. Theresults were clear. The autonomous lines grew equally well in CAPM basedon both types of D-MEM/F-12. The presence of the higher Fe (III) levelin CAPM based on standard D-MEM/F-12 had no effect. To confirm thatthese cell lines were androgen autonomous as defined by the loss ofsteroid and inhibitor growth regulation in CDE-serum, the next studieswere done. DU145 cells showed no inhibition of growth in 50% CDE-serum(FIG. 44). There was no androgenic effect whatsoever. A similar assaywith PC3 cells showed essentially the same results (FIG. 45). There wasno inhibition even in 50% CDE-horse serum, and no androgenic effect.Additionally, ALVA-41 cells are not iron sensitive (results not shown),and also are not sensitive to the serum-borne inhibitor (FIG. 46).

Discussion of Example 11

Together with the studies presented above, it appears that AR⁺ cells aresensitive to the serum-borne inhibitor, sensitive to the positiveeffects of steroid hormone and sensitive to Fe (III) inhibition. Incontrast, the DU145 and PC3 cells are insensitive to the serum-borneinhibitor, insensitive to the positive effects of androgen, andinsensitive to Fe (R. The results presented in this example continue todemonstrate the requirement for the action of a serum-borne mediator todemonstrate steroid hormone responsive cell growth in culture. Inaddition, autonomy may be the loss of the receptor for the serum factorand/or the loss of the intracellular steroid hormone receptor. If thishypothesis is correct it should be possible to identify cells thatpossess steroid receptors but still have lost “sensitivity” to thehormone by virtue of the lack of the effect of the inhibitor. Mostnotably, this is the case with DU145 and ALVA-41 cells. As defined byimmunohistochemistry, the DU145 cells are definitely AR⁺ (Brolin J etal. (1992) The Prostate 20, 281-295). As defined by a number ofcriteria, the ALVA-41 dells are AR⁺ (Nakhla A M and Rosner W (1994)Steroids 59, 586-589). A new concept explaining the progression ofnormal tissue cells to hormone autonomous cancers is provided herein anddiscussed in more detail in an Example below.

The use of CDE-serum is essential for the demonstration of androgen andother steroid hormone responsiveness in culture, but also limits theunderstanding of stimulatory or inhibitory roles of hormones or factorson prostate and other cancer cells because of the inclusion of anundetermined amount of undefined components. Serum-Free medium willcircumvent this problem.

In these studies, it is clear that exposure of androgen responsiveprostate cancer cells to Fe (III) results in cell death. Compoundscontaining available Fe (III) offer the possibility of new therapies forprostate cancer localized to the tissue. It is proposed that deprivationof iron will be a highly effective means of eliminating the mostdangerous hormone autonomous forms of prostate cancer. The mostimpressive growth requirement of hormone autonomous prostate and breastcancer cells is for diferric transferrin as a source of essential ironfor growth. Without this iron source, none of the epithelial cancer cellexamined could proliferate. In fact, within a two to three week periodall cells in the cultures were dead.

The measurement of thyroid hormone receptors in prostate cancer shouldbe initiated as a diagnostic tool to determine iron sensitivity.Moreover, a new therapy mode for tumors containing mixtures of bothhormone responsive and autonomous cells is suggested, based on theobservation that deprivation of iron can equally kill both types ofcancer. This suggests that systemic Fe (III) therapy for disseminatedprostate cancer may be efficacious. It is definitely possible that ironin the Fe (III) form and compounds containing it will be effectiveanti-prostate cancer treatments, and that direct injection (or painting)of localized prostate tumors or metastasis at other sites (e.g. bone)might effectively kill these cancers without concomitant systemiceffects. This therapy potentially could replace such protocols assystemic chemotherapy (physically damaging), radiotherapy (damage tocollateral tissues) or the use of locally acting radioactive gold chipsthat are complex to handle in the surgical environment and must beimplanted and removed surgically. Furthermore, iron therapies can berepeated frequently by application via transrectal or transurethralaccess, using conventional techniques. This approach is unique and hasnot been discussed or suggested anywhere else in the literature. Suchiron treatments may be a useful therapy for benign prostatic hypertrophy(BPH). As discussed above, this condition is very common in older menand is treated usually by surgery. Application of iron compounds is anew approach to treatment of BPH. Iron treatment also offers a uniqueapproach to the problem of residual breast cancer cells in mastectomysites or after lumpectomy. The present studies suggest that these sitesbe “painted”, injected or otherwise treated locally with a Fe(III)-containing solution to destroy residual early (ER⁺) breast cancercells not detected at surgery. Subsequent treatments of these sites byinjection can be used as follow-up therapy alone or with the currentadjuvant chemotherapy or radiation therapy common in lumpectomy treatedpatients.

Example 12 Growth in Serum-Free Defined Medium Versus Growth inCDE-Serum±E₂

Use of Defined Media to Verify the Presence of a Serum-Borne Inhibitor.

The defined media described in Example 9 were used to verify thepresence of a serum-borne inhibitor. The growth of six different ER⁺cell lines was compared in serum-free defined media (TABLE 6) to theeffects seen in cultures supplemented with CDE-horse serum. Thesestudies are shown in FIGS. 47 and 48. Estrogenic effects are recordedfor each set of conditions with each cell line.

MCF-7K Cells in Serum-Free and Serum Containing Medium±E₂.

The first studies were done with steroid hormone responsive human cancercell lines. FIG. 47A shows MCF-7K cell growth in serum-free DDM-2MF±10nM E₂. The population replicated logarithmically for 12 days. E₂ had noeffect on growth rate or saturation density. These results were incontrast to assays done in D-MEM/F-12 supplemented with CDE horse serum(FIG. 56B). Above 10% (v/v) serum, growth was progressively inhibited.The inhibition caused by any serum concentration was reversed by E₂.Measured on assay day 10, a 3 CPD estrogenic effect was observed whichwas a 2³ or 8-fold cell number increase. The experiments were also donewith MCF-7A cells with similar results (data not shown). This effect inCDE-serum was as great as that reported for a special response clone ofthe MCF-7 cell line (Wiese T E et al. (1992) In Vitro Cell Dev Biol 28A,595-602).

T47D Cells in Serum-Free and Serum Containing Medium±E₂.

FIG. 47C shows the growth of T47D cells in serum-free defined DDM-2MF±10nM E₂. Although a small effect of estrogen was observed on growth rate,the most significant effect was an increase in stationary densities by0.5 to 1.0 CPD. In contrast, the effect of E₂ was much greater in mediumcontaining CDE horse serum (FIG. 47D). At 50% (v/v) CDE-serum, growthwas completely inhibited. The estrogenic effect under these conditionswas >5 CPD. This was more than a 2⁵ or 32-fold hormone effect on cellnumber. Comparison of these results with those of others (Chalbos D etal (1982) J Clin Endocrinol Metab 55, 276-283; Schatz R W et al. (1985)J Cell Physiol 124, 386-390); Soto A M et al. (1986) Cancer Res 46,2271-2275; Soto A M and Sonnenschein C (1987) Endocr Rev 8, 44-52; ReeseC C et al. (1988) Ann NY Acad Sci 538, 112-121) confirmed that theconditions in FIG. 47D were substantially more effective. Comparableexperiments with the ZR-75-I line gave results intermediate betweenMCF-7 and T47D cells (data not shown). ZR-75-1 cells showed no effect ofE₂ in serum-free defined DDM-2MF. This line grows more slowly than MCF-7or T47D cells in defined medium and in serum-supplemented cultures(Ogasawara M and Sirbasku D A (1988) In Vitro Cell Dev Biol 24,911-920). The maximum estrogenic effects of the preferred embodimentrecorded with ZR-75-1 cells in D-MEM/F-12 with 50% (v/v) CDE-horse serumranged between 3 and 4 CPD after 14 days. This was greater than reportedby others in serum containing (Darbre P et al. (1983) Cancer Res 43,349-355; Kenney N J et al. (1993) J Cell Physiol 156, 497-514) or“serum-free” medium (Allegra J C and Lippman M E (1978) Cancer Res 38,3823-3829; Darbre P D et al. (1984) Cancer Res 44, 2790-2793).

LNCaP Cells in Serum-Free and Serum Containing Medium±E₂.

In another study, the effects of E₂ on the growth of the LNCaP humanprostatic carcinoma cell lines in defined medium and inserum-supplemented culture were compared. This cell line bears a pointmutation in the AR that permits high affinity binding of estrogens tothe altered receptor (Veldscholte J et al. (1990) Biochem Biophys ResCommun 173, 534-540; Veldscholte J et al. (1990) Biochim Biophys Acta1052, 187-194). In addition, it is possible that estrogens cause LNCaPgrowth via a separate functional ER (Castagnetta L A and Carruba G(1995) Ciba Found Symp 191, 269-286). Irrespective of mechanism,estrogens are known to promote LNCaP growth (Bélanger C et al. (1990)Ann NY Acad Sci 595, 399-402; Veldscholte J et al. (1990) BiochemBiophys Res Commun 173, 534-540; Veldscholte J et al. (1990) BiochimBiophys Acta 1052, 187-194; Castagnetta L A and Carruba G (1995) CibaFound Symp 191, 269-286). As presented herein (FIG. 47E), this cell linein serum-free defined CAPM showed essentially no E₂ effect on growthrate and ≦1.0 CPD on saturation density. When LNCaP growth assays weredone in medium with CDE-horse serum, the mitogenic effect of E₂ was >5CPD (FIG. 47F). Estrogenic effects herein were larger than reported byothers with LNCaP cells in serum containing culture (Bélanger C et al.(1990) Ann NY Acad Sci 595, 399-402; Castagnetta L A and Carruba G(1995) Ciba Found Symp 191, 269-286).

LNCaP Cell Growth in CAPM Defined Medium with CDE-Horse Serum and ±DHTor E₂.

To confirm that the serum-borne inhibitor can be assessed even in thepresence of all of the components of serum-free defined medium, anexample experiment is shown in FIG. 48. The LNCaP cells were grown inserum-free CAPM supplemented with increasing concentrations of CDE-horseserum without steroids and in assay dishes with the CDE-serum plus 10 nME₂ or 10 nM DHT. Without steroid, the CDE-horse serum showed theexpected progressive inhibition. Both the estrogen and androgen reversedthis inhibition completely at every serum concentration. Clearly, theinhibitor in serum possesses a very special quality that blocks theaction of the many mitogenic agents present in defined media.

GH₄C₁Cells in Serum-Free and Serum Containing Medium±E₂.

In the next studies, shown in FIG. 49, growth of rodent ER⁺ cell linesin defined medium and CDE serum-containing medium with and without E₂were compared. The study was with the GH₄C₁ rat pituitary tumor cellline. In serum-free PCM-9, E₂ had no effect on growth rate or saturationdensity (FIG. 49A). In contrast, the cells were highly estrogenresponsive in CDE-horse serum (FIG. 49B). In 30% (v/v) CDE-serum, theestrogenic effect was >4.5 CPD (i.e. >22-fold cell number increase). TheGH₄C₁ response obtained was substantially greater than that previouslyreported in cultures containing serum from a gelded horse (Amara J F andDannies P S (1983) Endocrinology 112, 1141-1143). Replicate studies withthe GH₁ and GH₃ rat pituitary tumor cells gave results equivalent tothose shown in FIGS. 49A and 49B (results not shown).

MTW9/PL2 Cells in Serum-Free and Serum Containing Medium±E₂. FIG. 49Cshows the effect of E₂ on growth of the MTW9/PL2 rat mammary tumor cellsin serum-free DDM-2A. There was a small effect on growth rate and a 1.0CPD effect on saturation density. When the same cells were assayed inD-MEM/F-12 containing CDE horse serum, the effect of E₂ was remarkable(FIG. 49D). Cell number differences of 2⁶ (i.e. 64-fold) were recordedin 50% (v/v) serum in a seven-day assay. This result agrees with thosepresented above in this disclosure. Furthermore, comparison of MTW9/PL2responses (FIG. 49D) to those of the human breast cancer cell responses(FIGS. 47B and 47D) confirms that the ER⁺ rat cells are the mostestrogen responsive mammary origin line yet developed.

H301 Cells in Serum-Free and Serum Containing Medium±E₂.

In the final studies, the effect of E₂ on the growth of the H301 hamsterkidney tumor cells in serum-free medium was compared to that in CDEhorse serum containing medium. Estrogen had no effect on H301 cellgrowth in serum-free defined CAPM (FIG. 49E). In contrast, E₂ inducedH301 cell number increases of >2⁴ (i.e. >16-fold) were recorded inD-MEM/F-12 containing ≧30% (v/v) CDE serum (FIG. 49F). The H301 responsewas similar to the MCF-7 cells in that 50% (v/v) CDE-serum did not fullyinhibit. The magnitude of the estrogenic effect with H301 cells wasequal to that reported by others studying this line in culturessupplemented with CDE serum prepared by different methods (Soto A M etal. (1988) Cancer Res 48, 3676-3680).

Discussion of Example 12

The serum-free defined medium provide a model system for identifyingphysiologically relevant new molecules. When completely serum-freedefined conditions were employed in the past, the effects of estrogenswere either marginal or insignificant as has been discussed above. Theearlier observations in completely serum-free defined culture mediumhave been extended in the present investigation. Direct comparisons weremade between estrogenic effects in serum-free defined culture andestrogenic effects in medium containing CDE serum. The results wereunequivocal. With every cell line tested, CDE serum was required todemonstrate significant estrogenic effects on logarithmic cell growthrates. A major advance provided was the clear demonstration that highconcentrations of serum are required to observe large magnitudeestrogenic effects. Furthermore, the inhibitory effects of serum aredose dependent even in the presence of the components used to formulateserum-free medium. This indicates that growth is progressivelynegatively regulated. This observation has physiological implications.Changes in the serum concentration of the inhibitor, or changes inavailability to target tissues, will have direct effects on the rate ofcell replication. The results in FIGS. 47 to 49 point to serum as thebest source yet identified to obtain the component that regulates sexsteroid responsive growth. The tissue origin of the serum regulatorremains to be investigated.

Example 13 Action of DES on Human AR⁺ LNCaP Prostate Cancer Cells

LNCaP Cells and DES Action.

Diethylstilbestrol (DES) is now used as one of the primary treatmentsfor prostatic cancer (Seidenfeld J et al. (2000) Ann Intern Med 132,566-577). Its action is likely mediated through thehypothalamus-pituitary axis (Seidenfeld J et al. (2000) Ann Intern Med132, 566-577). DES causes suppression of anterior pituitary hormones(e.g. LH and FSH) and therefore suppresses testicular output ofandrogens. Although it is thought that DES has no direct effects onprostate cancer cells, the development of the assay methodology set outherein permitted a direct assessment of this issue. The AR⁺ LNCaP cellswere used as a model for these tests (FIG. 50). As shown in FIG. 50A, 10nM DHT effectively reversed the inhibition caused by higherconcentrations of CDE-horse serum in D-MEM/F-12. Likewise, 10 nM E₂ alsoreversed the CDE-serum caused inhibition completely (FIG. 50B). However,the same concentration of DES was entirely ineffective (FIG. 50C). DESdid not reverse the serum caused inhibition. The synthetic estrogen hadno direct positive effect on LNCaP cell growth. In the final study ofthis series, DES addition to medium containing DHT or E₂ did not affectthe reversal caused by these two natural steroids (FIG. 50D). Therefore,DES is not a direct inhibitor of androgen or estrogen promoted LNCaPcell growth. The view that DES acts indirectly to cause chemicalcastration is consistent with the present results. These results aresupported by other studies indicating that DES does not bind to the ARof LNCaP cells (Montgomery B T et al. (1992) The Prostate 21, 63-73).

Discussion of Example 13

The fact that DES is a major treatment for prostate cancer but does notact directly on the tissue has therapeutic implications. For prostatecancer localized to the organ, or specific metastases in other locations(e.g. bone, liver or lung), direct application of Fe (III) offers atherapy with a different mode of action. It is also possible that localFe (III) therapy (as described in Example 12) can be used in conjunctionwith conventional systemic DES treatment to increase effectiveness abovethat with either treatment alone. There is another potential advantageof local Fe (III) treatment over systemic DES treatment. DES has manyside-effects in males. Some present considerable discomfort or medicalproblems. Locally applied Fe (III) is absorbed by the body to formnon-toxic mono ferric and diferric transferrin by chelation with thelarge pool of available apotransferrin. The iron containing proteinsformed are no problem for the body because they are the naturalphysiological forms of iron delivered to all tissues.

Example 14 Properties and Rationale for Serum Purification Source

Properties of the Serum-Borne Inhibitor(s).

It is clear from the results presented herein, and described inco-owned, concurrently filed U.S. patent application Ser. No. 09/852,958PCT/US2001/15183 entitled “Compositions and Methods for DemonstratingSecretory Immune System Regulation of Steroid Hormone Responsive CancerCell Growth,” which is hereby incorporated herein by reference, thatcharcoal-dextran treated serum contains a sex steroid hormone reversibleinhibitor(s) of target tumor cell growth in culture. This activity wasidentified as a progressive cell growth inhibition in culture mediumcontaining 10% to 50% (v/v) hormone depleted serum. Despite its firstproposal more than fifteen years ago, until the present invention, theinhibitor had yet to be purified, partially because of its instability.In an initial phase of investigations, a highly enriched fraction ofserum protein was produced whose estrogen reversible inhibitory activitywas stable and whose cell growth inhibitory effects replicate those seenwith full serum with a variety of sex steroid hormone target tumor celltypes in culture. Isolation was first attempted using an array ofstandard protein purification methods. Although they were expected toenhance stability, inhibitor activity was either not recovered after oneonly step or it was lost within two fractionation steps. In earlier work(Sirbasku D A et al. “Serum factor regulation of estrogen responsivemammary tumor cell growth.” Proceedings of the 1997 Meeting of the“Department of Defense Breast Cancer Research Program: An Era of Hope”,(Abstract) pp. 739-740, Washington, D.C., Oct. 31-Nov. 4, 1997)indicated that the inhibitor shared some properties with sex hormonebinding globulin (SHBG). These results were obtained with a purificationprotocol known to simultaneously yield purified corticosteroid bindingglobulin (CBG) and SHBG from human cord serum (Fernlund P and Lauren C-B(1981) J Steroid Biochem 14, 545-552). Additionally, it had beenobserved that the effect of calcium on both the estrogenic activity andthe binding of ³H-DHT to CDE-serum was remarkably similar to datapresented by others concerning the stability of human SHBG (Rosner W etal. (1974) Biochim Biophys Acta 351, 92-98). Different laboratories haveraised the issue of classical SHBG as the sex hormone reversibleinhibitor of target cell growth. That protein binds both androgens andestrogens in plasma and acts as a carrier system with cell signalingcharacteristics (Rosner W (1990) Endocr Rev 11, 80-91). However, in viewof the results presented herein and in U.S. patent application Ser. No.09/852,958 PCT/US2001/15183, SHBG was considered an unlikely candidatefor the inhibitor. Both CDE-horse serum and CDE-rat serum containconcentrations of inhibitor about equal to any of the other serum typesinvestigated but they do not contain SHBG (Corvol P and Bardin C W(1973) Biol Reprod 8, 277-282; Renior J-M et al. (1980) Proc Natl AcadSci USA 77, 4578-4582; Wenn R V et al. (1977) Endokrinologie 69,151-156). Nevertheless, rabbit anti-human SHBG purchased from AccurateChemicals not only immunoprecipitated the estrogenic activity inCDE-horse and rat serum, but also precipitated the ³H-DHT (i.e.SHBG-like) binding activity in these sera. This coincidence initiallyled to the mistaken conclusion that the inhibitor was SHBG-like(Sirbasku D A et al. “Serum factor regulation of estrogen responsivemammary tumor cell growth.” Proceedings of the 1997 Meeting of the“Department of Defense Breast Cancer Research Program: An Era of Hope”,(Abstract) pp. 739-740, Washington, D.C., Oct. 31-Nov. 4, 1997). Thismisconception turned out to be fortuitous, however, as it led to afurther exploration of the products obtained by the two-step cortisolagarose affinity and phenyl-Sepharose chromatography protocol. Thisprotocol, when used with horse and rat serum, provided material that atconcentrations of 10 to 15 μg/mL replicated the E₂ reversible inhibitioncaused by 30 to 50% (v/v) serum with steroid responsive human breastcancer cells, and responsive rat mammary, rat pituitary and Syrianhamster kidney tumor cells in culture. The inhibitor retained fullactivity for three years when stored unfrozen at −20° C. in the presenceof calcium, DHT and glycerol. As demonstrated herein, the long-standingproblem of inhibitor instability has been overcome, and a highly activepreparation became available to further probe molecular identity andmechanism(s) of action.

Mechanisms and Inhibitor Candidates.

The regulation estrogen target tissue cell growth has been a topic ofdynamic experimental interest beginning several years ago (Jensen E Vand DeSombre E R (1973) Science (Wash DC) 182, 126-134; O'Malley B W andMeans A R (1974) Science (Wash DC) 183, 610-620). Today, it is generallyaccepted that estrogen interaction with specific nuclear located DNAbinding receptors is necessary to initiate critical cell cycle events(Dickson R B and Stancel G M (1999) J Natl Cancer Inst Monograph No. 27,135-145). It is also highly likely that other non-steroid factors areessential participants in this process (Sirbasku D A (1978) Proc NatlAcad Sci USA 75, 3786-3790; Sirbasku D A (1981) Banbury Report 8,425-443; Dickson R B and Lippman (1987) Endocr Rev 8, 29-43; Soto A Mand Sonnenschein C (1987) Endocr Rev 8, 44-52). A number of years ago,studies were reported that indicated that serum-borne inhibitors, laternamed “estrocolyones”, had an important if not essential role (Soto A Mand Sonnenschein C (1985) J Steroid Biochem 23, 87-94; Soto A M andSonnenschein C (1987) Endocr Rev 8, 44-52). Estrocolyones were proposedto act as estrogen reversible inhibitors of steroid hormone targettissue cell growth. The results herein support this concept Over thecourse of several years, the inhibitor has been variously identified asan unstable M_(r) 70,000 to 80,000 protein (Soto A M et al. (1992) JSteroid Biochem Mol Biol 43, 703-712), the intact serum albumin molecule(Laursen I et al. (1990) Anticancer Res 10, 343-352; Sonnenschein C etal. (1996) J Steroid Biochem Mol Biol 59, 147-154), two domains of serumalbumin (Sonnenschein C et al. (1996) J Steroid Biochem Mol Biol 59,147-154) and SHBG (Reese C C et al. (1988) Ann NY Acad Sci 538,112-121). However, the roles of albumin and SHBG as estrogen relatedserum-borne growth regulators have been challenged (Sirbasku D A andMoreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 447-464;Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36,447-464; Soto A M et al. (1992) J Steroid Biochem Mol Biol 43, 703-712;Damassa D A et al. (1991) Endocrinology 129, 75-84). Prior to thepresent invention, no serum-derived inhibitor has been isolated, orotherwise identified at the molecular level, that replicates the largemagnitude estrogen reversible inhibitory effects of the presentlydisclosed inhibitors.

Discussion of Example 14 Purification of Source Serum

A goal of these studies was to obtain a high specific activitypreparation of the serum inhibitor and to define isolation and storageconditions that will permit its study over long experimental durations.Horse serum was selected for the initial studies because it had severaladventitious properties. First, it is a high content source of theestrogen reversible inhibitor that has biological activity with a broadrange of human and rodent sex steroid hormone target cells in culture.Second, when horse serum was steroid hormone depleted by charcoalextraction, the activity remained relatively stable at room temperaturefor a few weeks. Third, horse serum did not contain SHBG. This bypassedthe issue of classical M_(r) 94,000 dimeric SHBG as inhibitor.Additionally, horse serum is inexpensive, readily available, andpresented minimum biohazard during the application of the purificationprotocol.

Discovery Based on Serum Inhibitor Isolation.

The fact that the estrogen reversible inhibitory activity was ubiquitousin mammalian serum suggested that isolation from any one active specieswould lead to identification in the others, possibly withoutpurification. This is exactly what happened. The finalestrogen-reversible inhibitors isolated led to a major discovery ofphysiologic importance and revealed the first known link between thesecretory immune system and mucosal cancer development and growth.

Example 15 Cortisol Affinity and Phenyl Sepharose Isolation of the“SHBG-Like” Estrogen Reversible Inhibitor from CDE-Horse Serum

Outcome of the Search for the Estrogen Reversible Inhibitors.

As cited above, neither horse or rat serum contains SHBG. Therefore,these were the preferred sera to begin isolation. Partial purificationof the inhibitor from serum has been achieved initially by a two-stepprocedure. The partially purified inhibitor fractions are different thanthe serum derived inhibitor described in U.S. Pat. No. 4,859,585 (issuedto Sonnenschein and Soto), which has been more recently identified as asubtype domain of albumin. By contrast, IgA and IgM, preferably indimeric/polymeric form, are steroid hormone reversible inhibitors ofcell growth. The discovery of immune regulation of sex hormone dependentgrowth is unique.

Two-Step Cortisol-Agarose and Phenyl Sepharose Isolation Method.

Based on the perceived SHBG-like properties described above, a newapproach to the purification was taken. This method used a two-stepcortisol-agarose affinity and phenyl-Sepharose chromatography protocol.It had been employed by others to simultaneously yield purified humancord serum CBG and SHBG (Fernlund P and Laurell C-B (1981) J SteroidBiochem 14, 545-552). The method first required the synthesis of thecortisol affinity matrix. The cortisol-agarose affinity matrix wassynthesized and the initial purifications done as described (Fernlund Pand Laren C-B (1981) J Steroid Biochem 14, 545-552). An 80 mL bed volumecortisol-agarose column (2.5 cm×17.8 cm) was equilibrated with a buffercontaining 0.05 M piperazine, pH 5.5, with 0.2 M NaCl. Two liters ofhorse serum were charcoal-dextran extracted at 34° C. as describedabove. For two of the six preparations used in these studies, the serumwas depleted of steroid hormones by the Amberlite™ XAD-4™ resin method.There was no resulting difference in the purifications. After removing a30 mL sample for pre-column activity assay, the remaining volume wasadjusted to pH 5.5 with 1.0 N HCl. This was applied to the column at aflow rate of 30 to 40 mL per hour. Throughout the purification, the flowrates were maintained with a peristaltic pump. The effluent wascollected and a sample and adjusted to pH 7.2 for post-column assessmentof estrogen reversible inhibitory activity. After all of the serum hadbeen applied, the column was washed for 7 days at the same flow ratewith the equilibration buffer until the A_(280 nm) of the effluent was<0.06 versus water.

To recover the activity, the cortisol-agarose column was eluted with a500 mL linear gradient formed with 250 mL of the piperazine/NaCl bufferand 250 mL of the buffer with 1.0 mg/mL cortisol and 10% (v/v) methanol.After completion of the gradient, the column was washed with one volumeof the cortisol/methanol buffer. A total volume of 600 mL was collectedas 10 mL fractions. As reported by Fernlund & Laurell (Fernlund P andLaurell C-B (1981) J Steroid Biochem 14, 545-552), two separateA_(280 nm) or protein concentration ranges could be recognized, buttheir separation and individual chromatography on phenyl-Sepharose wasno more effective than pooling the entire 600 mL gradient elution andusing it for the next step. The total volume from the cortisol gradientwas reduced 5 to 8-fold by nitrogen gas pressure Amicon ultrafiltration(YM-10 membrane) and applied directly to the next column withoutdialysis or pH adjustment.

A 28 mL bed volume phenyl-Sepharose (1.5 cm×16 cm) was equilibrated with0.05 M Tris-HCl, pH 7.5, containing 0.5 M NaCl. The concentratedcortisol gradient volume was applied at a flow rate of 60 mL/hour (10 mLfractions). The first A_(280 nm) peak observed was a mixture of cortisoland CBG (Fernlund P and Laurell C-B (1981) J Steroid Biochem 14,545-552). These fractions were combined as cortisol affinity-phenylSepharose pool I (CA-PS-pool The column was then washed withequilibration buffer until the A_(280 nm) was reduced to 0.002 versuswater. The next buffer applied was 0.05 M Tris-HCl, pH 7.5 (60%, v/v)containing 40% (v/v) ethylene glycol. The A_(280 nm) peak observed withthis wash was combined to form CA-PS-pool II that corresponded to SHBGfrom human serum (Fernlund P and Laurell C-B (1981) J Steroid Biochem14, 545-552). The two pools were separately concentrated toapproximately 40 mL each and dialyzed separately against storage bufferwhich was 0.05 M Tris-HCl, pH 7.5, containing 0.15 NaCl, 0.05 M CaCl₂and 60% (v/v) glycerol. The dialysis further concentrated each sample.As last additions, 0.1 mM cortisol was added to CA-PS-pool I and 0.1 mMDHT was added to CS-PS-pool H. The pools were stored unfrozen at −20 C.Six replicate isolations were done. The protein yields ranged from 22.8to 37.7 for CA-PS-pool I and 5.82 to 12.2 mg for CA-PS-pool II. Based onan average of 60 grams of protein per two liters of CDE-horse serum(i.e. 30 mg/mL), CA-PS-pool II represented about 0.013% of the totalprotein in serum.

Cortisol Affinity and Phenyl Sepharose Isolation Results and SDS-PAGEMolecular Weight Estimation.

The chromatography profiles from the two-step cortisol affinity andphenyl Sepharose isolation of the inhibitor(s) activity from CDE-horseserum are shown in FIG. 51. The elution from phenyl Sepharose gave theCA-PS-pools I and II. CA-PS-pool I contained predominantly 58 kDa CBG(Rosner W and Bradlow H L (1971) J Clin Endocrinol Metab 33, 193-198) asconfirmed by SDS-PAGE and Western immunoblotting with rabbit anti-horseCBG as well as by partial amino acid sequencing of the first 10 to 20residues (results not presented). SDS-PAGE analyses of three examplepreparations of CA-PS-pool II are shown in FIG. 52A. Components of 67,58, 54, and 29 kDa were identified. These were compared to the 48 and 46kDa units identified for purified human SHBG (Khan M S et al. (1985)Steroids 45, 463-472) (FIG. 52A).

Native Molecular Weight Estimation.

Analyzes done under non-reducing and non-denaturing conditions usingSuperdex molecular sieve FPLC at neutral pH in buffers identifiedcomponents CA-PS-pool I in the exclusion volume at ≧900 kDa, andcomponents approximately 350 and 168 kDa (Sirbasku D A et al. “Serumfactor regulation of estrogen responsive mammary tumor cell growth.”Proceedings of the 1997 Meeting of the “Department of Defense BreastCancer Research Program: An Era of Hope”, (Abstract) pp. 739-740,Washington, D.C., Oct. 31-Nov. 4, 1997). Comparison of the results fromdenaturing and non-denaturing conditions confirmed that the CA-PS-poolII was still heterogeneous and that the activity was most likely asubunit containing high molecular weight protein(s).

Removal of Storage Solution Components Before Bioassay.

Before conducting bioassays of the inhibitory activity in thephenyl-Sepharose pools, the glycerol and steroid hormones in the storagebuffers were removed. If DHT is not removed completely from CA-PS-poolII, the inhibitory activity was substantially diminished or eliminatedentirely. Samples (0.5 to 15 mL) were introduced into Slide-A-Lyzer®(Pierce) cassettes of molecular weight cutoff 10,000. The cassettes wereincubate twice with stirring in two liters of Tris-HCl, pH 7.4,containing 10 mM CaCl₂ for four hours at 34° C. to remove excess freesteroids and glycerol. Next, the cassettes were transferred to the samebuffer containing 20% (v/v) of a charcoal-dextran mixture prepared asdescribed above. After 18 hours at 37° C., the cassettes weretransferred to another two-liter volume of the same buffer containing10% (v/v) of the charcoal-dextran mixture and dialysis continued withstirring for another 6 to 8 hours. Finally, the cassettes were rinsedlightly with water and the dialyzed material recovered according tomanufacturers instructions. The contents were sterilized by 0.2-μm-poremembrane filtration and stored at 4° C. These preparations were usuallyused within a few weeks.

Assay of CA-PS-Pool I Estrogen Reversible Inhibitory Activity withMTW9/PL2 Cells.

When assayed with MTW9/PL2 cells, CA-PS-pool I contained 20 to 25% ofthe units of estrogen reversible inhibitory activity recovered from thephenyl Sepharose column (data not shown). With two preparations notpresented, the cortisol gradient pool shown in FIG. 51 was made 1.5 MNaCl before application to the phenyl Sepharose column equilibrated atthe same higher salt concentration. Under these conditions, theCA-PS-pool I contained >90% CBG, as estimated by SDS-PAGE, but showedeither no estrogen reversible activity or only traces (results notpresented). Irrespective of the ionic strength or pH of the cortisolaffinity pool applied to phenyl Sepharose, ethylene glycol was requiredto elute the majority of the activity.

Assay of CA-PS-Pool II Estrogen Reversible Inhibitory Activity withSeveral ER⁺ Cell Lines.

Despite method variations with phenyl Sepharose, CA-PS-pool II alwayscontained ≧75% of the activity recovered. In a crucial test ofsignificance, CA-PS-pool II was assayed to determine if it replaced theeffects of CDE-serum with eight different ER⁺ cell lines. The resultsare shown in FIG. 53. The estrogen reversible inhibitory effects ofCA-PS-pool II were investigated with five rodent tumor cell linesderived from three different estrogen target tissue tumors, and threeseparate estrogen sensitive human breast cancer cell lines. The cellswere added to medium with 2.5% (v/v) CDE-horse serum plus increasingconcentrations of CA-PS-pool II±10 nM E₂. The first lines evaluated werethe GH₁, GH₃, and GH₄C₁ rat pituitary tumor cells (FIGS. 53A, 53B and53C, respectively). They were chosen first because these lines are wellknown for hormone regulation of differentiated tissue specific functionsin culture and exceptional sensitivity to a variety of hormonesincluding estrogens (Tashjian A H Jr (1979) Methods Enzymol 58, 527-535;Haug E and Gautvik K M (1976) Endocrinology 99, 1482-1489; Haug E (1979)Endocrinology 104, 429-437; Amara J F and Dannies P S (1983)Endocrinology 112, 1141-1143). At 10 μg/mL, CA-PS-pool II was fullyinhibitory with all three GH lines. Growth was reduced to near seeddensity levels (i.e. <0.5 CPD). By this measure, >1,700-fold increase inpotency had been achieved versus full CDE-serum. The ED₅₀ with the GHcells was 6 to 8 μg/mL which was a 300 to 800-fold specific activityincrease compared to full serum. E₂ reversed the effects of theCA-PS-pool II at every inhibitory concentration. CA-PS-pool II replacedthe effects of full CDE-serum with these cells. FIGS. 53D and 53E showsimilar experiments with the estrogen sensitive H301 hamster kidneytumor cells and the MTW9/PL2 rat mammary cells, respectively. CA-PS-poolII was most inhibitory at 15 μg/mL with both lines. The ED₅₀ were in therange of 5 to 10 μg/mL. As with the GH lines, E₂ completely reversed theeffects of the inhibitor. Again, CA-PS-pool II replaced the effects offull CDE-serum with these cells. With human breast cancer cell linesMCF-7K, ZR-75-1 and T47D, the results were similar (FIGS. 53F, 53G, and53H, respectively). Addition of 10 to 15 μg/mL of CA-PS-pool II causedmaximum inhibition. The ED₅₀ concentrations were 6 to 9 μg/mL. As withER⁺ rodent cell lines, E₂ completely reversed the inhibition caused byCA-PS-pool II. Again, CA-PS-pool II replaced the effects of fullCDE-serum with these cells.

Cortisol-Agarose Affinity Removal of the Inhibitor from CDE-Serum.

Next it was determined if the cortisol affinity chromatography had notremoved the majority of the activity from serum. To test this, threecell lines were analyzed with pre- and post cortisol column samples.FIGS. 54A and 54B show the effect of a single column passage on theinhibitory activity for T47D human breast cells. The ED₅₀ of thepre-column CDE-serum was 7% (v/v) (FIG. 54A). Post-column, even 50%(v/v) serum did not achieve ED₅₀ (FIG. 54B). FIGS. 54C and 54D show thesame studies with the GH₃ rat pituitary cells. In this case, a singlecolumn passage completely depleted the activity. Complete depletion wasalso observed with the H301 hamster kidney cell line (FIGS. 54E and54F).

Storage Conditions and SHBG Related Properties.

At completion of the two-step isolation, the pools were stored in thepresence of sufficient glycerol to prevent freezing at −20° C. Inexperiments not shown, the estrogen reversible inhibitor wasprogressively less stable without addition of glycerol, calcium and/orsteroid hormone. Dialysis against buffers without calcium is mostdefinitely to be avoided. Freeze/thaw is very harmful, even with calciumand DHT present. Assays of −20° C. glycerol stored CA-PS-pool II over atwo year period indicated no decay in activity. Clearly, the storageconditions known to stabilize functional SHBG (Fernlund P and Lauren C-B(1981) J Steroid Biochem 14, 545-552; Rosner W et al. (1974) BiochimBiophys Acta 351, 92-98) also favored retention of estrogen reversibleinhibitor activity in CA-PS-pool II.

Labeled Steroid Hormone Binding to CA-PS-Pool I.

CA-PS-pool I was determined to contain CBG by criteria cited above.Additionally, this pool was examined by Scatchard analysis for bindingof tritium labeled steroid hormones. The results are summarized in TABLE9. The association constants (K_(a)) of the labeled hormones showed theorder cortisol>progesterone>>>sex steroid hormones. The K_(a) ofcortisol binding at 34° C. was 1.41×10⁹M⁻¹ that was equal to that ofnative rat CBG when analyzed at 4° C. (Rosner W (1990) Endocr Rev 11,80-91). However, it was higher than the K_(a) of 5.2×10⁷ M⁻¹ for humanCBG measured at 23° C. (Rosner W and Bradlow H L (1971) J ClinEndocrinol Metab 33, 193-198). The binding characteristics of steroidsto CBG from several species have been studied (Rosner W (1972) J SteroidBiochem 3, 531-542). The similarity of the results herein furthersupports the conclusion that CA-PS-pool I contains predominantly CBG.

Labeled Steroid Hormone Binding to CA-PS-Pool II.

The estrogen reversible inhibitor activity in CDE-serum correlated withthe binding of tritium labeled sex steroid hormones. This suggested arelationship between the estrogen reversible inhibitor and SHBG.However, the K_(a) for ³H-DHT binding to CDE-serum at 34° C. was3.90×10⁷ M⁻¹. However, it is important to note that this was at least 20times lower than that of purified human SHBG at 0.99×10⁹M⁻¹ for DHT or2.2×10⁸ M⁻¹ for E₂ at 37° C. (Rosner W and Smith R N (1975) Biochemistry14, 4813-4820). To determine if CA-PS-pool II possessed the same sexhormone binding properties as whole CDE-serum, and/or human SHBG, thenext study was conducted. Scatchard analysis of ³H-DHT binding toCA-PS-pool II was done at 34° C. The estimated K_(a) was 5.88×10⁷ M⁻¹.Replicates (N=3) gave a K_(a) range 4.5−10×10⁷M⁻¹. Computer analysisindicated a single class of binding sites although correlationcoefficients were approximately 0.7. Similar analyses were done with³H-E₂, ³H-progesterone and ³H-cortisol. The results with all fourlabeled steroids are summarized in TABLE 7. The K_(a) order wasDHT>E₂>>>cortisol>progesterone. The K_(a) for sex steroid hormonebinding to the CA-PS-pool II was similar to whole CDE-serum but 20 to50-fold lower than human SHBG.

Table 7

TABLE 7 Summary of the Scatchard Analysis of phenyl-Sepharose pools Iand II with four labeled steroid hormones Steroid Hormone CA-PS-Pool ICA-PS-Pool II (³H-labeled) K_(d) (M) K_(a) (M⁻¹) K_(d) (M) K_(a) (M⁻¹)Cortisol 7.10 × 10⁻¹⁰ 1.41 × 10⁹ 1.89 × 10⁻⁶ 5.30 × 10⁵ Progesterone1.70 × 10⁻⁹ 5.90 × 10⁸ 7.89 × 10⁻⁶ 1.17 × 10⁵ 17β-estradiol 1.05 × 10⁻⁵9.51 × 10⁴ 2.83 × 10⁻⁸ 3.55 × 10⁷ Dihydrotestosterone 6.05 × 10⁻⁶ 1.64 ×10⁵ 1.43 × 10⁻⁸ 6.99 × 10⁷

Western Immunoblotting with Anti-Human SHBG.

The above shows that the estrogen reversible inhibitor sharedimmunological properties with human SHBG. To investigate further,Western immunoblotting of CA-PS-pool II was done with anti-human SHBG.The results are presented in FIG. 52B. Western analysis with theanti-SHBG recognized the same four components seen with Coomassie Bluestaining in FIG. 52A. These same four components have also beenidentified with whole CDE-serum using Western analysis with anti-humanSHBG (data not shown). In Western immunoblotting studies not presented,anti-human SHBG did not identify horse serum albumin. This confirmedthat the 67 kDa Coomassie Blue stained component present in theCA-PS-pool II was not 68 kDa horse serum albumin. These results providedadditional support for the conclusion that albumin is not the estrogenreversible inhibitor activity of serum. These results also very clearlydemonstrated that the SHBG used to raise antibodies in rabbit had notbeen purified to homogeneity, but rather had been used at a more “crude”state. (In a personal communication, it was also confirmed by themanufacturer of the anti-SHBG antibody that the SHBG fraction used forantibody production was not highly purified and had not been sizefractionated.)

Discussion of Example 15

There has been one very critical problem with the estrocolyonehypothesis. Estrocolyone has never been purified and shown to act asdescribed (Soto A M and Sonnenschein C (1987) Endocr Rev 8, 44-52). Theactive pool isolated from the two-step procedure (i.e. CA-PS-pool II)certainly does not bind steroid hormones with sufficient affinity to actas estrocolyones (TABLE 7). Growth is activated at picomolarconcentrations while the affinity (Kd) of E₂ with CA-Pool II is about10⁻⁸M. This discrepancy is simply far too large to accept the role ofestrogens in growth as binding the inhibitor and thereby preventing itsaction on target cells (Soto A M and Sonnenschein C (1987) Endocr Rev 8,44-52). The fact that proteins in CA-PS-pool II bind steroids is notgermane to the mechanism of action of these hormones in growthregulation under physiological conditions.

The results of steroid hormone binding may however be germane to the useof high dose treatments of breast cancer. Care must be taken whenconsidering that high doses of estrogen, androgen, progesterone andcortisol all have the potential for binding the active agent inCA-PS-pool II and therefore may reduce the effective concentration ofinhibitor. The assays described in this Example can be applied tobiological fluids and plasma to determine if steroid concentrations areexcessive and to evaluate proper levels with changes in treatmentregimes.

The results presented herein indicate that the proposed new model ofcell growth is a favored mechanism. Steroid hormones appear to act aspositive agents via internal high affinity receptors (e.g. ERγ) whereasserum-borne inhibitors act at the surface to block growth. Thecombination of the two signals dictates cell proliferation rates. Thisdata further supports the assertion that the ERγ can be used fordiagnostic purposes in ER⁺ cancers, preferably in the same way thatconventional ER receptor screening is now performed.

A highly enriched fraction of serum protein was prepared whose estrogenreversible inhibitory activity is stable and whose effects replicatethose seen with full serum with a variety of sex steroid hormone targettumor cell types in culture. Because early studies mistakenly indicatedthat the inhibitor shared various properties with SHBG, a two-stepcortisol-agarose affinity and phenyl-Sepharose chromatography protocolwas applied. A highly enriched “SHBG-like” preparation was obtained. At10 to 15 μg/mL, it replicated the E₂ reversible inhibition caused by 30to 50% (v/v) serum with steroid responsive human breast cancer cells,and responsive rat mammary, rat pituitary and Syrian hamster kidneytumor cells in culture. The inhibitor retained full activity for morethan one year when stored unfrozen at −20° C. in the presence ofcalcium, dihydrotestosterone and glycerol. This study demonstrated thatthe longstanding problem of inhibitor stability has been overcome andthat a high specific activity preparation was now available to furtherprobe molecular identity. These results clearly differentiate thisinhibitor preparation from any previously described type of estrogenreversible inhibitor (i.e. estrocolyone). Moreover, no previousinhibitor composition, at a concentration ≦15 μg/mL, can supplant theeffects of full serum to give estrogenic effects ≧3 CPD with several ER⁺cell lines from different tissues and different species.

The most active inhibitor preparation obtained in this study appeared tohave multiple components present. The separation and identification ofthese components would yield additional assays and preferred reagentsand methodologies for testing new hormone-like and anti-hormone likesubstances. The results in FIG. 52 suggest that there may be more thanone inhibitor. The active serum-derived inhibitor fraction can be useddirectly in tests of new compounds, substances, mixtures andpreparations from natural and synthetic sources to estimate bothestrogenic and androgenic activity in culture. Large-scale preparationof this purified serum fraction is possible by using larger affinitycolumns and proportionately increased serum volumes, similar to existingtechnology employed for purifying other biological products. It isadvantageous that only, small quantities of the purified serum fractionare needed for cell growth

Example 16 Serum-Free Assay Systems for Measuring Large MagnitudeSteroid Hormone Mitogenic Responses with the Two-Step Purified Inhibitor

The above-described studies with several different sex steroid sensitivecell lines demonstrated that the effects of a partially purifiedestrogen reversible inhibitor could readily be assayed in the presenceof a low concentration (i.e. 2.5%) of CDE-serum. The next step was toeliminate the serum completely and to show estrogen responsiveness underfar more defined conditions.

Second Analysis of Serum-Free Growth±E2.

Experiments were conducted using completely serum-free medium, and themagnitude of the estrogenic effects observed in defined medium was againcompared to those seen in medium containing CDE-serum. ER⁺ tumor cellgrowth was measured first in serum-free defined culture±10 nM E₂.Similar experiments have been reported in FIGS. 47 and 48. The newassays were included here because the first experiments were done twoyears earlier. The results show the stability of the cell lines used andthe fact that serum-free defined medium is highly reproducible. Morerecent results are shown with the MCF-7K human breast cancer cells (FIG.55A), the T47D human breast cancer cells (FIG. 55B), the GH₄C₁ ratpituitary tumor cells (FIG. 55C), and the H301 Syrian hamster kidneytumor cells (FIG. 55D). All four-cell lines grew logarithmically forseveral days in defined and reached densities of 0.5 to 1.0×10⁶ cellsper 35-mm dish. The media formulations were based on standard D-MEM/F-12as described in TABLE 6. Growth rates were optimized to 70% or more ofD-MEM/F-12 containing 10% (v/v) fetal bovine serum. The resultspresented in FIG. 55 show little or no E₂ effect on growth in definedmedium. Barnes and Sato (Barnes D and Sato G (1980) Nature (Lond) 281,388-389) have reported similar negative results with another strain ofMCF-7 cells in a different formulation of defined medium. Consideringthe variety of cell types assayed herein, the present results and theresults of others, the lack of estrogenic effects in serum-free definedmedium was not related to chemical composition of any one medium nor wasthere a major problem with time dependent variation of cell lineproperties.

Effects of CDE-Serum on ER⁺ Cells in Different Formulations ofSerum-Free Defined Medium.

The experiments in FIG. 56 were done to show that serum could be addeddifferent formulations of defined medium and still cause estrogenreversible inhibition. Effects are shown with CDE-horse serum±10 nM E₂and T47D cells DDM-2MF (FIG. 56A), MTW9/PL2 cells in DDM-2A (FIG. 56B)and GH₄C₁ cells in PCM-9 (FIG. 56C). Definitely, the serum-borneinhibitor(s) was fully effective in three different formulations ofdefined medium and with three different estrogen target tissue celltypes.

Effects of CA-PS-Pool II on ER⁺ Cell Growth in Serum-Free DefinedMedium.

The estrogen reversible inhibitory effects of CA-PS-pool II wereexamined with eight ER⁺ cell lines growing in different serum-freedefined media (FIG. 57). The cell lines were the MCF-7K cells (FIG.57A), the T47D cells (FIG. 57B), the ZR-75-1 human breast cancer cells(ATCC) (FIG. 57C), the GH₁ (ATCC) (FIG. 57D), GH₃ (ATCC) (FIG. 57E), andGH₄C₁ (FIG. 57F) rat pituitary tumor cells, the MTW9/PL2 rat mammarytumor cells (FIG. 57G), and the H301 Syrian hamster kidney tumor cells(FIG. 57H). At 20 to 30 μg/mL, this fraction completely inhibitedgrowth. The inhibition was totally reversed by 10 nM E₂. The E₂ effectson cell number were in the range from 33 to 72-fold (i.e. CPD=2^(5.04)to 2^(6.18)). The activity was not replaced by serum albumin at 5 mg/mL(data not shown). The estrogen mitogenic effects seen in defined mediumcontaining only a few μg/mL of protein were equal to or greater thanthose seen in medium containing 30 to 50% (v/v) CDE-horse serum withevery ER⁺ cell line tested (TABLE 8). Plainly, the serum-free conditionsestablished herein are the most defined model assay systems yetestablished to demonstrate estrogen responsiveness in vitro.

TABLE 8 Summary of the Maximum Estrogenic Effects in D-MEM/F-12 plusCDE-horse Serum 10 nM E₂ versus those in Serum-free Defined MediumSupplemented with CA-PS-pool II MAXIMUM ESTROGENIC EFFECTS IN SERUM-FREE MAXIMUM ESTROGENIC MEDIUM PLUS CELL LINES EFFECTS IN CDE-SERUMCA-PS-POOL II MCF-7K 3.40 CPD (2^(3.40) = 10.5-fold) 5.84 CPD (2^(5.84)= 57.3-fold) T47D 5.38 CPD (2^(5.38) = 41.6-fold) 5.88 CPD (2^(5.88) =58.9-fold) ZR-75-1 3.84 CPD (2^(3.84) = 14.3-fold) 5.21 CPD (2^(5.21) =37.0-fold) GH₁ 4.71 CPD (2^(4.71) = 26.2-fold) 5.04 CPD (2^(5.04) =32.9-fold) GH₃ 4.78 CPD (2^(4.78) = 27.4-fold) 5.04 CPD (2^(5.04) =32.9-fold) GH₄C₁ 4.82 CPD (2^(4.82) = 28.2-fold) 5.11 CPD (2^(5.11) =34.5-fold) MTW9/PL2 6.22 CPD (2^(6.22) = 74.5-fold) 6.18 CPD (2^(6.18) =72.5-fold) H301 4.33 CPD (2^(4.33) = 20.1-fold) 6.01 CPD (2^(6.01) =64.4-fold) CPD (2^(CPD) = Fold Cell Number Increases Above ControlsWithout Estrogen)

Discussion of Example 16

The studies presented in FIG. 57 and TABLE 8 summarized unequivocally,and for the very first time, demonstrate that large magnitude estrogenmitogenic responses can be observed in completely serum-free definedmedia containing 2 mg/mL total protein. Furthermore, the responses shownin FIG. 57 either equal or exceed others previously observed inpartially serum-free media with ZR-75-1 human breast cancer cells(Allegra J C and Lippman M E (1978) Cancer Res 38, 3823-3829; Darbre P Det al. (1984) Cancer Res 44, 2790-2793) or with a variety of otherestrogen sensitive (ER') human and rodent cell lines in medium withhormone depleted or deficient serum (Amara J F and Dannies P S (1983)Endocrinology 112, 1141-1143; Natoli C et al. (1983) Breast Cancer ResTreat 3, 23-32; Soto A M et al. (1986) Cancer Res 46, 2271-2275; Wiese TE et al. (1992) In Vitro Cell Dev Biol 28A, 595-602).

These results have a number of important implications, one of which isthat they support the aspect of the estrocolyone hypothesis (Soto A Mand Sonnenschein C (1987) Endocr Rev 8, 44-52) that relates to thepresence in serum of a meaningful inhibitor(s). Also, in view of thepresent results, there is no doubt that the inhibitor(s) is/arecompletely estrogen reversible. However, the present experimentalresults do not confirm that the steroid hormones interact with theinhibitor with sufficient affinity to support that aspect of theestrocolyone hypothesis. The results in TABLE 7 indicate that thissteroid hormone binding aspect of the estrocolyone hypothesis is highlyunlikely.

The estrogen reversibility of the inhibitor with every target cell typestudied under the rigorous conditions of serum-free defined culturesuggests physiologic relevance. The large magnitude of the effects is astrong statement in favor of significance. This is especially clear whenconsidering the fact that the first experiments with 30 to 50% (v/v)serum contained 15 to 25 mg/mL of protein, whereas the later tests usingserum-free medium required only 20 μg/mL of isolated protein.

The active fraction isolated from horse serum represented only 0.01 to0.04% (w/w) of the total protein. Nonetheless, it effectively regulatedeight ER⁺ cell lines derived from three species and three differenttarget tissues. These observations are evidence that a broadlyapplicable serum fraction has been identified. Furthermore, theserum-free medium results suggest that a common agent(s) maycoordinately regulate estrogen responsive tissue growth in vivo and thatthe concept of estrogen reversible negative control may be far-reaching.The results support the conclusion that in vitro studies can be used toidentify important new aspects of in vivo endocrine physiology. Theresults of the cell growth experiments in defined medium have manypractical applications. It has been demonstrated herein that a modelcell growth assay system now exists that is valuable for assessing awide variety of cell growth effects.

Cells in serum-free medium grow in response to nutrients, growthfactors, metal delivery proteins, adhesion proteins, and various classesof hormones. All of these components are mitogenic in the sense thatthey contribute to cell replication. Nonetheless, the addition of only20 μg/mL of inhibitor to block growth completely bears directly on thequestion of the progression of normal steroid target cells to fullyhormone autonomous cancers. The inhibitor preparation used herein hasthe properties of a family of tissue regulators first named “chalones”.These proposed cell regulators are water-soluble and tissue specific(but not species specific) proliferation inhibitors that are reversibleby physiologic stimuli including hormones (Bullough W S (1975) Life Sci16, 323-330; Finkler N and Acker P (1978) Mt Sinai J Med 45, 258-264).The studies presented herein support this classic concept as it appliesto sex steroid hormone target tissues. The molecular identification ofthe serum inhibitor(s) promises not only to further support the role ofestrogens as “necessary”, but also to establish that “chalone-like”entities likely are the missing “sufficient” components that account forestrogen regulation of tissue growth. The application of serum-freedefined medium conditions along with the use of a high specific activityfraction to demonstrate estrogen responsiveness in culture is unique. Itshould be noted that “chalones” have never before been identified. Theresults presented herein indicate, and in U.S. patent application Ser.No. 09/852,958 PCT/US2001/15183 entitled “Compositions and Methods forDemonstrating Secretory Immune System Regulation of Steroid HormoneResponsive Cancer Cell Growth,” hereby incorporated herein by reference,that the immune system is the long sought after source of these tissuespecific inhibitors. In the series of studies described herein, thetissues are the mucosal tissues.

Example 17 Chemical and Immunological Properties of the PartiallyPurified CA-PS-Pool II Inhibitors and Identification as IgA and IgM

This Example describes chemical and physical confirmation that thesought-after serum-borne cancer cell growth inhibitor(s) include atleast IgA and IgM.

Antibodies Against the CA-PS-Pool II Components.

Preparative SDS-PAGE was done on the CA-PS-pool II fraction, and afterlocalization of the 54 kDa band, the 54 kDa band was eluted and preparedfor rabbit antibody production by HTI (Ramona, Calif.). The antibodiesraised were very potent and reacted with CA-PS-pool II (FIG. 58). Theydid not cross react with CBG (CA-PS-pool I). However, despite greatcare, it was evident that the anti-54 kDa was raised against a mixtureof 67, 58 and 54 kDa subunits (FIG. 58). The reaction was definitelystrongest with the 54 kDa component, but clearly identifiable with the67 kDa and 58 kDa bands as well. This apparent problem turned out to bean advantage, and allowed positive identification of the active agentsin CA-PS-pool II. It was investigated whether the activity in CA-PS-poolII might have been isolated because of affinity for the agarose matrixrather than as a consequence of the steroid hormone ligand attached toagarose, noting from interpretation of unrelated studies, that agarosealone can bind immunoglobulins and give SDS-PAGE bands at 67, 58 and 54kDa. Therefore, it was thought possible that IgG was the estrogenreversible inhibitor.

Antibodies Against the 54 kDa Component of CA-PS-Pool II and Blocking ofthe Estrogen Reversible Inhibitor Activity.

Based on the results in FIG. 58, it was apparent that the 54 kDaantiserum might be used to determine if the biological activity residedin any of the 67, 58 or 54 kDa bands. The next study was done to resolvethis important issue. The results were pivotal. FIG. 59 shows that thepurified material in CA-PS-pool II was completely inhibitory at 20 to 40μg/mL. Addition of even a 1:5000 dilution of anti-54 kDa blocked theeffect of the inhibitor. In control studies, rabbit pre-immune serum hadno effect even at 1:100 a dilution (data not shown). It was evident thatanti-54 kDa serum contained the antibody to the activity.

Anti-54 kDa Serum Recognizes Authentic Horse IgA, IgM and IgG.

Next, authentic horse IgA was obtained from Accurate Chemicals, andhorse IgM was obtained from Accurate Chemicals and Custom MonoclonalInternational. The material from Custom Monoclonals was custom purifiedby an affinity method with a monoclonal antibody against horse IgM Fcand further purified by molecular sieve chromatography to be sure ofelimination of other immunoglobulins (a common problem). IgGs wereobtained from Zymed (San Francisco, Calif.), Sigma (St. Louis, Mo.) orThe Binding Site (San Diego, Calif.). The Western analysis shown in FIG.60 demonstrates these results. The results show clear cross-reactionwith 67 kDa IgM heavy chain, 58 kDa IgA heavy chain and 54 kDa IgG heavychain but no reaction with horse albumin.

Assay of Estrogenic Effects Controlled by Commercially Purchased HorseIgG, IgA and IgM in 2.5% CDE-Horse Serum with MTW9/PL2 Cells.

FIG. 61 demonstrates that at concentrations up to 59 μg/mL, horse IgGdid not cause inhibition of MTW9/PL2 cell growth in 2.5% CDE-horseserum. There was no significant estrogenic effect caused by IgG. FIG. 62shows very clearly that commercially prepared horse serum derived IgM(Custom Monoclonals), was very active. At concentrations of 20 to 50μg/mL, IgM completely inhibited the growth of the MTW9/PL2 cells (i.e.<1.0 CPD). Addition of 10 nM E₂ reversed the inhibition nearlycompletely. Estrogenic effects of 4 to 5 CPD were seen (FIG. 62). FIG.63 shows the same general results with commercially prepared horse serumderived IgA (Accurate). The only apparent difference was that IgA wasslightly more effective than IgM. These results clearly proved that theactive components in CA-PS-pool II were IgA and IgM. This was a clearsequence of studies culminating in evidence supporting IgA and IgM. Thatthese immunoglobulins would prove to be the inhibitor was completelyunexpected. Although these two active classes of immunoglobulins (IgAand IgM) are well-established secretory products of normal breast cells,there was no previous suggestion in the prior art that they play a rolein the negative regulation of estrogen-dependent cell growth. Theseimmunoglobulins are major proteins in milk whose hormone-related localproduction in breast tissue is well documented, and their function inthe body's secretory immune system is well known.

Alternate Methods of Obtaining Horse Serum IgG, IgM and IgA.

IgG can be purified using a Hytrap matrix, which is a mixture ofimmobilized Protein A and Protein G, employing a technique described byothers (Lindmark R et al. (1983) J. Immunol. Meth 62, 1-13; Kronvall Get al. (1969) J Immunol 103, 828-833; Akerstrom B et al. (1986) J BiolChem 261, 10240-10247). IgM can be obtained using a mannan bindingprotein isolation method normally applied with human serum (Nevens J Ret al. (1992) J Chrom 597, 247-256). However, yields are low. Anothermethod based on anti-IgM immunoglobulins linked covalently to Sepharoseis far more effective. This same procedure with immobilized anti-IgAimmunoglobulins can be used to isolate IgA (Tharakan J In: AntibodyTechniques, Malik V S & Lillehoj E P, Eds, 1994, Academic, Press, SanDiego, Calif., Chapter 15). Horse IgA can also be purified using animmobilized Jacalin lectin method usually reserved for human samples(Roque-Barreira M C et al. (1986) Braz J Med Biol Res 19, 149-157).However, it can be modified for non-human species. The buffers aremodified to contain 10 to 50 mM CaCl₂ to bind IgA from other species.Even then, yields are not high. The preferred methods for horse IgA andIgM use immobilized antibodies.

Purification of Rat Serum Immunoglobulins.

Three isolations of the estrogen reversible inhibitor from separateone-liter batches of adult rat serum were conducted. This was done fortwo important reasons. First, the estrogen reversible activity in alltypes of adult serum, including rat, were assayed with a highly estrogensensitive MTW9/PL2 rat mammary tumor cell line. It was useful to confirmthe horse serum purification results with a homologous experimentalsystem. Second, the confirmation that rat IgA and IgM regulated ratmammary tumor cell growth would open the possibility of combined testingof new therapeutic substances both in vitro and in vivo. To summarize,the same “CBG” and “SHBG” fractions were obtained from rat serum by themethods of Fernlund & Laurell as had been obtained from horse serum. Thechromatography profiles of the rat separations (not presented) were verysimilar to those presented in FIG. 51. The only major difference wasthat with rat serum, the first peak (i.e. CA-PS-pool I) contained noCBG. At pH 5.5, rat CBG did not significantly bind to the affinitymatrix. Rat serum CA-PS-pool I and CA-PS-pool II both contained only twoCoomassie Blue stained bands when analyzed by SDS-PAGE (FIG. 64A). Thesewere approximately 55 kDa and 54 kDa. They were somewhat lower molecularweights than found with horse, and there were fewer bands. To test ifeither rat band was IgG, a Western analysis was performed with rabbitanti-rat IgG (FIG. 64B). The antibody did not recognize the Coomassiestained bands but did react with control IgG. However, when examinedwith very specific heavy chain monoclonal antibodies raised to rat IgG1,IgA, and IgM (purchased from Zymed), the Western analysis was clear(FIG. 65). Both the commercially purified rat immunoglobulins (purchasedfrom Zymed) and the two-step purified pools showed cross-reaction withanti-IgA (weakly), anti-IgG1 subtype (strong reaction) and anti-IgM(moderate reaction) (FIGS. 65A, 65B, 65C, respectively).

Rat and Horse Serum Active Pools Isolated by the Two-Step Procedure ofFernlund and Laurell have the Same Classes of Immunoglobulins.

The same classes of immunoglobulins obtained by the two-step procedureof Fernlund and Laurell (Fernlund P and Laurell C-B (1981) J SteroidBiochem 14, 545-552) with horse serum were found when rat serum was thestarting material. This was considered to be further confirmation thatbinding to the agarose matrix was more important than to the immobilizedcortisol. It should be noted that in the original Fernlund and Laurellreport using human cord serum does not address possible immunoglobulincontamination, however (Fernlund P and Laurell C-B (1981) J SteroidBiochem 14; 545-552). This is particularly curious because humanimmunoglobulins bind to agarose (Smith R L and Griffin C A (1985)Thombosis Res 37, 91-101).

Labeled Steroid Hormone Binding to the “SHBG-Like” Pools from Rat Serum.

As described in TABLE 6, CA-PS-pool II from horse serum binds sexsteroids with an affinity of about 10⁻⁸ M. This same Scatchard analysiswas done with an active fraction from rat serum. TABLE 9 shows theresults of these studies with four labeled steroid hormones. It is clearthat sex steroid hormones bind with a higher affinity than progesteroneor cortisol. The binding affinities of rat and horse preparations werevery similar. In both cases, the affinities tend to rule out theestrocolyone hypothesis because it requires E₂ binding in the picomolarrange.

TABLE 9 Summary of the Scatchard Analysis of the “SHBG-like” Pools fromRat Serum with Labeled Steroid Hormones Steroid Hormone CA-PS-Pool II(3H-labeled) K_(d) (M) K_(a) (M⁻¹) Cortisol 5.7 × 10⁻⁶ 1.8 × 10⁵Progesterone 6.9 × 10⁻⁶ 1.4 × 10⁵ 17β-estradiol 4.1 × 10⁻⁸ 2.4 × 10⁷Dihydrotestosterone 2.4 × 10⁻⁸ 4.1 × 10⁷

Evaluation of the Rabbit Anti-SHBG Cross-Reaction with the Active Poolsfrom the Two-Step Isolation of Fernlund and Laurell.

As shown above in FIG. 52B, Western analysis with the anti-SHBG detectedhorse IgA, IgM and IgG. Additionally, anti-SHBG immunoprecipitated theestrogenic activity of horse serum (results not presented): To extendthese results, it was established that rabbit anti-human SHBG recognizeda number of the major classes and subclasses of rat immunoglobulins.SDS-PAGE with Coomassie blue staining (FIG. 66A) was compared toidentification of the same proteins by Western analysis with anti-SHBG(FIG. 66B). These results leave very little doubt that the human plasmaderived SHBG used to raise antibodies in rabbits was not homogeneous butin fact was a “crude” preparation contaminated with severalimmunoglobulins.

Test of Rat IgG, IgA and IgM for Estrogen Reversible Inhibitory Activitywith MTW9/PL2 Rat Mammary Tumor Cells.

All of the rat immunoglobulins described in this section were purchasedfrom Zymed as the highest quality available. Their activity was assessedwith MTW9/PL2 cells in 2.5% (v/v) CDE-rat serum, as described above. Theactivity of rat IgG (all subclasses combined) was assessed (FIG. 67).There was no inhibitory effect at up to 50 μg/mL. Rat IgA was a potentestrogen reversible inhibitor (FIG. 68). At 20 to 50 μg/mL, itcompletely inhibited growth. Addition of 10 nM E₂ completely reversedthe inhibition. The estrogenic effects recorded were >5 CPD. The resultswith rat IgM were very similar (FIG. 69). At 20 to 50 μg/mL, itcompletely inhibited growth. Addition of 10 nM E₂ reversed theinhibition. The estrogenic effects recorded were >5 CPD. It is essentialto note that IgA or IgM replaced the effect of full CDE-rat serum withMTW9/PL2 cells. With a completely homologous system (i.e. cell line,basal 2.5% CDE-serum, and immunoglobulins), the results were clear. IgAand IgM were the sought after serum-borne inhibitors from rat.

Discussion of Example 17

The identification of IgA and IgM as serum-borne inhibitors fullyseparates these inhibitors from the teachings of U.S. Pat. Nos.4,859,585 (Sonnenschein) and 5,135,849 (Soto), which arrived at nomolecular identification of the inhibitor. The series of investigationsdescribed above demonstrate that a very longstanding problem has beensolved. While the solution is significant, an even more an importantconsequence of this knowledge is the fact that for the very first time,mucosal cell hormone dependent growth has been linked to a naturalimmune regulation. Moreover, this information has direct application tothe diagnosis, genetic screening, prevention and therapy of breast andprostate cancer and a high likelihood of applications to other mucosalcancers, as also described elsewhere herein.

During the purification of both the horse serum and the rat serumestrogen reversible activity, SUPERDEX™ (Pharmacia) molecular sievechromatography of the final mixtures indicated the presence of <20% 160kDa monomeric immunoglobulins. The majority of the material was of muchlarger mass. Because IgA exists naturally as monomer, dimer andpolymers, there was a question concerning which of these is/areinhibitory form(s). The SUPERDEX™ results strongly favor thedimer/polymer form. This was confirmed also with commercially preparedIgA that was obtained from hybridoma and myeloma cell lines. The IgAfrom these was >80% dimer/polymer. It was very active as an inhibitor.In light of these results, it is suggested that these forms are the“good” type of IgA in the body, and that direct measurement of theirconcentration in plasma and body fluids has diagnostic and prognosticapplications.

Test methods similar to those described above, but performed with adefined, preferably minimum serum, plus purified immunoglobulininhibitor (“inhibitor spiked serum”) provide a new approach toevaluating potentially cell growth affecting substances, mixtures andcompounds that might be influenced by serum components. For example, aserum composition might contain steroid hormone free serum, such as astandard, commercially available fetal bovine serum preparation, and apredetermined amount of an immunoglobulin inhibitor, i.e., one or moreof IgA, IgM or IgG. Testing under these conditions, with a known amountof inhibitor in the serum, may be desirable or required when thesubstance has potential for inactivation/activation by a serum componentor when it has lipophilic properties that require a minimum proteinconcentration in the medium to prevent loss.

Another valuable application of the immunoglobulin inhibitors will be inidentifying substances that may have direct effects on the action of theimmunoglobulins to cause inactivation. An assay of this nature is uniquein the sense that incubation of substances with the immunoglobulin canbe done before the assay to determine effects on natural immuneresponses. Changes in environmental/chemical factors that affect thebody's immune system are of major medical concern. They also are ofgreat concern to veterinary medicine. Chemicals/nutritional supplementsmay affect immune function of domestic animals and thereby affect humanfood supplies.

This series of investigations demonstrate at least two immunoglobulininhibitors in serum. More than one inhibitor was suggested by theconventional purification data in a preceding Example, and was provedtrue in succeeding examples. There may still be other useful estrogenreversible immunoglobulin inhibitors in serum that are yet to beidentified from serum or tissue sources. The methods described in thisExample have direct application to the search for new compounds thatmimic the effect of the immunoglobulins as estrogen reversibleinhibitors. Such application opens a new avenue of search for anticancerdrugs.

Example 18 Regulation of Steroid Hormone-Responsive and ThyroidHormone-Responsive Cancer Cell Growth in Serum-Free Defined Medium bySecretory and Plasma Forms of IgA and Plasma and Cell Culture DerivedIgM

The determination of whether purified IgA and IgM from several speciesmimicked the sex steroid hormone reversible inhibitors isolated fromhorse in serum was sought. These studies included ER⁺ tumor cellsderived from rodents as well ER⁺ and AR⁺ cells from human cancers.Completely serum-free defined culture conditions were used to performcell growth assays using the purified inhibitors. The total proteinconcentration in the media was <2 mg/mL. The estrogenic and androgeniceffects observed in these assays are unique, as like effects have notbeen achieved previously in completely serum-free defined medium.

Sources of Purified IgA and IgM.

Human IgM was purified from human plasma as described using immobilizedmannan-binding protein (Nevens J R et al. (1992) J Chromatography 597,247-256). As an example of the effectiveness of this isolation, FIG. 70shows SDS-PAGE and Coomassie Blue Staining with two preparations ofhuman plasma IgM prepared. Human IgA1 and IgA2 were purified usingimmobilized Jacalin (Roque-Barreira M C and Campos-Neto A (1985) JImmunol 134, 1740-1743; Kondoh H et al. (1986) J Immunol Methods 88,171-173; Pack T D (1999) American Biotechnology Laboratory 17, 16-19;Loomes L M et al. (1991) J Immunol Methods 141, 209-218). Rat IgA andIgM were purchased from Zymed. The effectiveness of the Jacalin methodwith human plasma is shown in FIG. 71. Horse IgA and IgM were purchasedfrom Accurate, Sigma and Custom Monoclonals. IgA and IgM from otherspecies or as products from cell culture are purchased from Sigma orAccurate. Human IgA and IgM were bought also from Sigma and Accurate.Human secretory (milk) IgA (sIgA) was purchased from Sigma or Accurate.

MTW9/PL2 Rat Mammary Tumor Cells.

For this series of experiments the serum-free defined medium was thepreferred formulation of DDM-2A described in TABLE 6. The cell growthassays with this cell line in DDM-2A testing increasing concentrationsof human plasma IgM is shown in FIG. 72. Human plasma IgM completelyinhibited growth by 20 to 60 μg/mL. The ED₅₀ was about 12 μg/mL. Basedon an IgM M_(r) of 950,000, the ED₅₀ concentration was 1.3×10⁻⁸ M.Complete inhibition was at 2.2×10⁻⁸ M. These concentrations arecertainly within the physiological range of IgM in the plasma and bodyfluids such as breast milk. Based on these studies, a comparison wasdone in completely serum-free defined DDM-2A medium of the effects of 40μg/mL of rat plasma IgA±E₂, rat plasma IgM±E₂, and horse plasma IgM±E₂(FIG. 73, expressed as (A) cell numbers and (B) CPD). From the CPDcalculations it was clear that no matter the species source, IgA and IgMwere very potent estrogen reversible inhibitors of MTW9/PL2 cell growth.

One problem occurred with the MTW9/PL2 cell assays that initially causedconcern. Human IgA was purchased from Sigma as the milk derivedimmunoglobulin. It was far less expensive than plasma IgA. For reasonsthat at first were not clear, this material was at best only partiallyinhibitory and often not inhibitory. As will be discussed below with GH₁cells, this turned out to be a significant clue to the mechanism ofaction of the immunoglobulins. Nonetheless, it is known that the heavychains of IgM and IgA from different species share primary structurehomology. This is not true of the variable regions of the light chains.The results presented support the possibility of Fc-like receptormediation of the IgA and IgM effects on MTW9/PL2 cells.

GH₁, GH₃ and GH₄C₁ Rat Pituitary Tumor Cells.

For this series of experiments the serum-free defined medium was thepreferred formulation of PCM-9 described in TABLE 6. The next serum-freedefined medium studies were done with GH₁ cells. Example assays areshown. This cell line was highly estrogen responsive in the presence ofhomologous rat myeloma derived IgA (FIG. 74). Maximum estrogenic effectwas >5 CPD or more than a 32-fold estrogen-induced increase in cellnumber in 10 days. A similar assay with human plasma derived IgA showednearly the same results (FIG. 75). Indeed, human IgA showed greaterinhibition at 10 μg/mL. Another study with human IgM demonstrated thatit was also an estrogen reversible inhibitor of GH₁ cell growth (FIG.76). It was not as inhibitory as IgA with this cell line, but certainlystill effective. As discussed above, in the Background of the Invention,during the secretion process a fragment of about 80% of the poly-Igreceptor (including the five extracellular domains) becomes attached tothe dimeric/polymeric form of IgA to form secretory IgA or sIgA. Thereceptor fragment is called the “secretory component”. After secretion,sIgA can be readily isolated from human milk. The effect of milk derivedsecretory IgA (sIgA) was evaluated with the GH₁ cells in PCM-9, and theresults of a representative study are shown in FIG. 77. These resultswere strikingly different than those obtained with plasma derived IgA(pIgA). SIgA was not inhibitory even at 20 μg/mL. Considering why thetwo different forms of IgA behaved so differently in the GH₁ cells, thepoly-Ig receptor was recognized as a potential candidate for themediator of the action of IgA/IgM. The poly-Ig receptor has not beenpreviously associated with any growth related function. The poly-Igreceptor is concerned with process of transcytosis of IgA/IgM, asconceptually illustrated in (FIG. 78). SIgA already has the receptorbound in the sense of the secretory piece in association with the Fcdomains of the dimer. FIG. 79 illustrates schematically the structuresof inactive monomeric IgA, the connecting or joining “J” chain, thestructure of the active dimer with “J” chain, the secretory piece orsecretory component, and the dimeric IgA structure plus secretorycomponent attached, as generally understood. The illustration shows thatthe Fc domains of dimeric IgA are blocked by the secretorypiece/component. Access to the Fc domains is required for binding to thepoly-Ig receptor.

The present series of cell growth assays above were continued with therelated GH₃ cells, again in serum-free defined the preferred formulationof PCM-9 medium. Rat myeloma derived IgA was an effective estrogenreversible inhibitor of these cells in a 9 day growth assay (FIG. 80).The maximum estrogenic effect exceeded 5. CPD. A similar assay with ratIgM was conducted (FIG. 81). It showed even greater inhibition at 10μg/mL than with IgA. The estrogenic effect recorded in 10 days wasnearly 6 CPD. These same assays were next repeated with the humanimmunoglobulins. Human pIgA was an estrogen reversible inhibitor of GH₃cell growth (FIG. 82). It was not as effective as its rat counterpart,but the estrogenic effect with the human immunoglobulin was still 4 CPD.Also, human IgM was effective with GH₃ cells (FIG. 83). Again theestrogenic effect was about 4 CPD. In the final study with GH₃ cells, itwas again apparent that human milk derived sIgA was not inhibitory (FIG.84).

The studies above with GH₁ and GH₃ cells were continued with the relatedGH₄C₁ line, again in serum-free defined PCM-9 medium. Rat myelomaderived IgA was an effective estrogen reversible inhibitor of thesecells in a 9 day growth assay (FIG. 85). The maximum estrogenic effectapproached 5 CPD. A similar assay with rat plasma IgM was conducted(FIG. 86). It showed slightly less inhibition than IgA. The estrogeniceffect recorded in 10 days was nearly 4 CPD. These same assays were nextrepeated with the human immunoglobulins. Human pIgA was an estrogenreversible inhibitor of GH₄C₁ cell growth (FIG. 87). It was not aseffective as its rat counterpart, but the estrogenic effect with thehuman immunoglobulin was still almost 4 CPD. Also, human pIgM waseffective with GH₄C₁ cells (FIG. 88). The estrogenic effect was about 5CPD. In the final study with GH₄C₁ cells it was again apparent thathuman milk derived sIgA was not inhibitory (FIG. 89).

H301 Syrian Hamster Kidney Tumor Cells.

The studies with this cell line were done in the preferred formulationof CAPM defined medium described in TABLE 6. Because hamster IgA and IgMwere not available, these experiments began with plasma IgA from mouse(FIG. 90). Mouse IgA was very effective with hamster H301 cells. Theestrogenic effect was >5 CPD. Human plasma IgA was also effective (FIG.91A). The maximum estrogenic effect reached 4 CPD. Secretory IgA wasinactive (FIG. 91B). With this cell line, human IgM also was an estrogenreversible inhibitor. As shown in FIG. 92, a dose-response studydemonstrated that in serum-free defined medium with 40 μg/mL of humanplasma IgM, concentrations of 0.1 to 1.0 picomolar E₂ caused significantgrowth (p<0.01). This data demonstrate the extraordinary sensitivity ofthe serum-free defined cell growth assays in the presence ofimmunoglobulin. The data in FIG. 92 provide strong support for the viewthat the H301 cells can be used to characterize the new ERγ proposed inthis disclosure. Further description of the rationale and evidence for anew growth regulation very high affinity estrogen receptor, ERγ, isfound in a following Example.

MCF-7A and MCF-7K Human Breast Cancer Cells.

For this series of experiments the serum-free defined medium was thepreferred formulation of DDM-2MF described in TABLE 6. Two highlyapplied MCF-7 human breast cancer cell strains were applicable to thisseries of investigations. As shown with MCF-7A cells in DDM-2MFserum-free defined medium, plasma IgA was highly effective as anestrogen reversible inhibitor. The estrogenic effect exceeded 4 CPD in10 days (FIG. 93A). In contrast, sIgA was inactive (FIG. 93B). With theMCF-7K strain, the results were nearly identical. Plasma IgA waseffective (FIG. 94A) and sIgA was inactive (FIG. 94B). The estrogeniceffects caused by pIgA were replicated by substitution of plasma IgM.With MCF-7A and MCF-7K, pIgM was an effective estrogen reversiblesustaining estrogenic effects of >4 CPD (FIGS. 95 and 96, respectively).In a final study of this series, an E₂ dose-response experiment wasconducted with MCF-7K cells in DDM-2MF plus 40 μg/mL of plasma IgM. Theresults were remarkable. Estrogen at as low as 0.1 picomolar caused morethan one-half maximum growth response (FIG. 97). The extraordinarysensitivity of this assay methodology is clearly established. Theseresults add more evidence that a very high affinity estrogen receptor(i.e. ERγ) regulates growth and is yet to be defined in human breastcancer cells.

T47D Human Breast Cancer Cells.

The T47D cell line was assayed for immunoglobulin effects in thepreferred formulation of serum-free defined medium DDM-2MF described inTABLE 6. As shown in FIG. 98A, human plasma IgA was a very effectiveestrogen reversible inhibitor with T47D cells. The maximum estrogeniceffect was 6 CPD or a 72-fold cell number increase in 12 days. Incontrast, sIgA was inactive at up to 20 μg/mL (FIG. 98B). Likewise,human plasma IgM is effective (FIG. 99), demonstrating completeinhibition of cell growth by 20 μg/mL IgM. The estrogenic effect was 5CPD in 12 days. In experiments not shown, the effects of plasma derivedIgM were compared to myeloma derived IgM. This study yielded the sameestrogenic effects with both sources of IgM. Again, the antigenicdeterminant appears to be unimportant. The results support the view thatthe heavy chains dictate the activity. In other studies with T47D cellsin defined medium containing 40 μg/mL, the dose-response effects with E₂showed more than one-half maximum growth at 0.1 picomolar (FIG. 100).These results continue to fortify the theme that the methods describedin this Example allow investigation of potential estrogenic compoundsand substances that might be present in samples of industrial orbiological materials at very low concentrations. It is also apparentthat the data supports the view that a high affinity ERγ regulatesgrowth.

ZR-75-1 Human Breast Cancer Cells.

For these experiments the serum-free medium was the preferredformulation of DDM-2MF described in TABLE 6. Plasma IgA was an estrogenreversible inhibitor with ZR-75-1 cells (FIG. 101A). The estrogeniceffect was recorded at 5 CPD in 14 days. As seen before with the otherER⁺ cell lines above, sIgA was not an inhibitor with ZR-75-1 cells (FIG.101B). Plasma IgM was also assayed with the ZR-75-1 cells (FIG. 102). Itwas a potent estrogen reversible inhibitor under these completelyserum-free defined conditions. As discussed above, this line had beenthought to be estrogen responsive in serum-free culture. However, theformer methods were not serum-free. As disclosed herein, it has now beenestablished in entirely different culture conditions and shown that thisline is truly estrogen growth responsive in culture.

HT-29 Human Colon Cancer Cells.

For this series of experiments the serum-free defined medium was thepreferred formulation of CAPM described in TABLE 6. As expected fromendocrine physiology, colon is not a sex steroid hormone growthregulated tissue as are others such as breast, uterus, ovary andpituitary. However, it was discovered that this tissue is thyroidhormone growth responsive. As shown in FIG. 103, HT-29 human coloniccarcinoma cells grow in CAPM independently of the presence of thyroidhormone. This growth is promoted by the other factors present in CAPMminus T₃. However addition of plasma IgM at 40 μg/mL had a dramaticeffect. In the absence of T₃ HT-29 cell growth was inhibited to ≦1.0 CPDin 10 days. Addition of increasing concentrations of T₃ restored growth(FIG. 103). This demonstrates that colonic cancer cells respond tothyroid hormones in the same manner that ER⁺ cells respond to E₂.Estrogens and thyroid hormones belong to the same superfamily ofreceptors and both are required for normal physiologic growth anddevelopment (Williams G R and Franklyn J A (1994) Baillieres ClinEndocinol Metab 8, 241-266; Tsai M J and O'Malley B W (1994) Annu RevBiochem 63, 451-486). This is the first demonstration of a secretoryimmunoglobulin acting directly as a thyroid hormone reversible growthinhibitor of a human origin colon cancer cell line.

LNCaP Human Prostatic Carcinoma Cells.

For this series of experiments the serum-free defined medium was thepreferred formulation of CAPM described in TABLE 6. LNCaP cells werenegatively regulated by plasma IgA (FIG. 104A). The immunoglobulin was aDHT reversible inhibitor that was completely effective at 10 μg/mL. Theandrogenic effect was >5 CPD in 12 days. As seen with the ER⁺ cell linesabove, sIgA was not inhibitory with LNCaP cells (FIG. 104B). Twodifferent types of human IgM were also compared with LNCaP cells (FIG.105). They were plasma derived and myeloma derived IgM. Despite thedifferences in antigen binding domains, both forms were equallyinhibitory and both forms were reversed by 10 nM DHT. These resultsindicate that the Fe/heavy chain of IgM is the functional activator ofthe inhibition.

Summary of the Estrogenic Effects of IgM on ER⁺ Cell Growth.

FIG. 137 presents a summary of the effects of IgM derived from differentspecies with a variety of ER⁺ cell lines. This summary presents themaximum estrogenic effects recorded under conditions described above inserum-free defined medium with each cell line±10 nM E₂. Estrogeniceffects ranged from 4 to >7 CPD. Comparison of the results in FIG. 106with those in TABLE 8 show in general that the results achieved incompletely defined medium are equal to or greater than those seen inCDE-serum cultures.

Discussion of Example 18

These methods will permit evaluation of industrial, environmental,biological, medical, veterinary medicine and other potential sources ofestrogenic or androgenic activity under the most sensitive conditionsyet developed. Estrogenic activity is measurable at ≦1.0 picomolarconcentrations. Two cell lines, MTW9/PL2 and H301, are preferredpotential sources of identification of the new growth regulatory ERγ.The evidence presented with MCF-7 and T47D human breast cancer cellssupport the presence of a new growth regulatory ERγ. The serum-freemethods described herein provide unique tools to search for ERγ. Assaysconducted under these conditions permit estimation of estrogensensitivities in ranges not approachable by other technology. Thesemethods can also be adapted to measurement of the inhibitor inbiological fluids available in only small supply. For example, coupledwith use of XAD-4™ resin extraction to remove steroids, bodily fluidsand other source materials can be assayed on small scale to determinethe concentration of effective inhibitor. This is of particular interestbecause IgA in plasma is >90% inactive monomer and <10% activedimer/polymer. Measurement of IgA by conventional methods gives totalconcentrations, and does not determine the concentration/presence ofactive inhibitor. The present biological activity method has distinctfeatures and advantages, and can serve as an adjunct measurement.

The serum-free defined medium assays described herein can be used tosearch for new compounds that mimic the action of immunoglobulins toblock cancer cell growth in its early stages. This screening can be doneunder conditions in which serum proteins might interfere. Compoundsso-identified can next be evaluated by addition of CDE-serum or XAD-4™treated serum to determine if serum proteins interfere and to determinedrug efficacy in vitro under both serum-free defined medium conditionsand serum supplemented conditions. Serum-free defined medium methods canbe used for screening of compounds that may either enhance or inhibitimmune function at the epithelial cell level. Compounds with theseactivities may have utility as immune enhancers to help reduce the riskof cancer development. These assay methods offer a screening tool forsuch compounds that has not been available before. Larger magnitudeeffects permit greater accuracy with the new assay methods whenestimating effects of substances that are less potent than naturalestrogens.

Example 19 A New High Estrogen Affinity Growth Regulating EstrogenReceptor (ERγ)

This Example provides evidence of a never before recognized receptorthat mediates estrogen responsive cell growth, and discusses potentialapplications for the receptor as a diagnostic and prognostic tool.

Steroid Hormone Superfamily of Receptors.

Estrogens, androgens, progestins, corticosteroids, mineral steroids,vitamin D, retinoic acid and thyroid hormone receptors all belong to afamily of DNA binding intracellular receptors that are activated bybinding of the appropriate hormone/ligand (Evans R M (1988) Science(Wash DC) 240, 889-895; Giguere V (1990) Genetic Eng (NY) 12, 183-200;Williams G R and Franklyn J A (1994) Baillieres Clin Endocrinol Metab 8,241-266; Kumar R and Thompson E B (1999) Steroids 64, 310-319; Pemrick SM et al. (1994) Leukemia 8, 1797-806; Carson-Jurica M A et al. (1990),Endocr Rev 11, 201-220; Tsai M J and O'Malley B W (1994) Annu RevBiochem 63, 451-486; Alberts B et al. (1994) Molecular Biology of TheCell, 3rd edition, Garland Publishing, New York, pp 729-731). Theestrogen receptor described in the citations above is now designated theclassical estrogen receptor alpha (ERα). Its role in steroid regulatedgene expression has been studied extensively and often reviewed(Yamamoto K R (1985) Annu Rev Genet. 19, 209-252; Green S and Chambon P(1991) In: Nuclear Hormone Receptors, Academic Press, New York, pp15-38; Tsai M-J and O'Malley B W (1994) Annu Rev Biochem 63, 451-486;McDonnell D P et al. (1992) Proc Natl Acad Sci USA 89, 10563-10567;Landel C C et al. (1994) Mol Endocrinol 8, 1407-1419; Landers J P andSpelsberg T C (1992) Crit. Rev Eukary Gene Exp 2, 19-63; Cavailles V etal. (1994) Proc Natl Acad (Sci USA 91, 10009-10013; Halachmi S et al.(1994) Science (Wash DC) 264, 1455-1458; Brasch K and Ochs R L (1995)Int rev Cyto 159, 161-194; Härd T and Gustafsson J-Å (1993) Acc Chem Res26, 644-650).

Human Mutation and Mouse Knock-Out Studies of ERα and ERβ.

It is noteworthy that estrogen resistance in man is caused by a mutationin the ERα (Smith E P et al. N Eng J Med 331, 1056-1061). The moststartling fact is that this point mutation (i.e. cytosine→thymidine)generated a premature stop codon, but was not lethal. Although manymetabolic abnormalities were noted, development into adulthood wasobserved without expression of a functional ERα. This fact is furtherstrengthened by the experiments with ERα gene knockout mice (Couse J Fand Korach K S (1999) Endocr Rev 20, 358-417). Those authors state “thelist of unpredictable phenotypes in the α ERKO (estrogen receptorknockout) must begin with the observation that generation of an animallacking a functional ERα gene was successful and produced animals ofboth sexes that exhibit a life span comparable to wild-type”.Furthermore, in the review of the ERKO results it was not possible toconclude that the ERα, regulated estrogen responsive cell growth.Indeed, functions normally ascribed to the ERα seemed unaffected. Infact, only development in tissues such as breast seemed best correlated(Boccchinfuso W P and Korach K S (1997) J Mammary Gland Biol Neoplasia2, 323-334). The situation with ERKO mice and ERβ is similar (Couse J Fand Korach K S (1999) Endocr Rev 20, 358-417). The results from ERβknockout suggest an indirect role of this receptor via stromal tissue(Gustafsson J-Å and Warner M (2000) J Steroid Biochem Mol Biol 74,254-248). Certainly a direct growth role for ERβ in breast epithelialcells was not established. The results available from ERKO do not yetprovide confidence that either the ERα or the ERβ mediate estrogenresponsive cell growth.

ERα and Growth Regulation.

There are other pertinent lines of evidence that relate to the role ofthe ERα and growth. The first is from a study of transfection ofestrogen receptor negative cells with the full length functional ERα(Zajchowski D A et al. (1993) Cancer Res 53, 5004-5011). Theinvestigators arrived at a remarkable result. They had expected toregain estrogen responsive growth in the transfected hormone independentcells. This was definitely not the case. Instead, addition of E₂ causedcell growth inhibition. The results indicated that ERα was not apositive mediator, but instead a negative regulator. However, similarlytransfected estrogen responsive cell lines such as MCF-7 and T47D werenot E₂ inhibited.

As previously mentioned herein, considering the results of the presentinvestigations, it is concluded that another positive acting ER existsin the MCF-7 and T47D cells and that its function is dominant andsustains growth related gene expression even with the inhibitory ERαpresent. The existence of two ER receptors is also indicated in an olderstudy of the growth of the GH₄C₁ rat pituitary tumor cells in culture(Amara J F and Dannies P S (1983) Endocrinology 112, 1141-1143). Thoseinvestigators demonstrated a biphasic effect of E₂ on these cells. Atpicomolar concentrations, E₂ caused growth. At higher concentrations, E₂induced prolactin production secretion and inhibited growth. If tworeceptors are operating, the growth receptor is more sensitive to E₂whereas the ER regulating gene expression (e.g. prolactin mRNAproduction) is activated by higher concentrations of estrogen. This samebiphasic action of estrogen on the growth of T47D human breast cancerscells has also been noted (Chalbos D et al. (1982) J Clin EndocrinolMetab 55, 276-283). Low concentrations promoted growth, whereas higherlevels were inhibitory. Indeed, a biphasic effect also was noted withthe MCF-7 cell line (Soto A M and Sonnenschein C (1985) J SteroidBiochem 23, 87-94). When this observation is coupled with the clearstatements of Soto et al (Soto A M et al. (1986) Cancer Res 46,2271-2275) that “the free estradiol levels needed for maximum responseare significantly lower than estrophilin (i.e. ERα) K_(d)s”, there isfurther support for the view that an ER exists that regulates growth andis more estrogen sensitive (i.e. lower K_(d)) than the classical ERα.While those investigators conclude that the results exclusivelysupported their estrocolyone hypothesis, and excluded ERα as thepositive growth regulator, there was no recognition of the possibilityof a much higher affinity receptor different than ERα. Finally, there isone other issue that has perplexed endocrinologists and cancerbiologists for several years. Breast cancer is sometimes treatable withhigh doses of estrogen (Segaloff A (1981) Banbury Report 8, 229-239). Ifthe ERα is the only growth mediator, one is forced into many otherpostulates to explain this observation (Reese C C et al. (1988) Ann NYAcad Sci 538, 112-121). Indeed, it may be that full occupation of ERα isinhibitory and that another receptor is the positive signal. One otherissue that is of special interest with regard to the ERα is the factthat many tissues are known to express ERα but are not growth responsiveto estrogen. Instead, estrogens cause tissue specific gene expression.Considering the results of the present investigations, it is proposedthat those tissues lack ERγ, and are therefore not growth responsive.

Variant Estrogen Receptors.

Certain variant estrogen receptors have been identified recently byothers. For example, from the estrogen growth responsive T47D humanbreast cancer cell line, there have been three isoforms of the ERαidentified in one study (Wang Y and Miksicek R J (1991) Mol Endocrinol5, 1707-1715) and another three in a different study (Graham M L et al(1990) Cancer Res 50, 6208-6217). With another two estrogen growthresponsive human breast cancer cells lines, the MCF-7 and ZR-75-1,another ERα variant was identified that lacked the entire exon 4 of thereceptor (Pfeffer U et al. (1993) Cancer Res 53, 741-743). Variantreceptors have also been identified from human breast cancer biopsyspecimens (Murphy L C and Dotzlaw H (1989) Mol Endocrinol 3, 687-693).Another truncated variant of ERα acts as a natural inhibitor of theaction of the wild-type ERα (i.e. unchanged receptor) (Fuqua S A et al.(1992) Cancer Res 52, 483-486). Another type of variant has receivedwide attention because it has constitutive transcriptional activitywithout the steroid hormone ligand bound (Fuqua S A et al. (1991) CancerRes 51, 105-109). Even normal human breast epithelial cells show severalnatural variants of ERα (Yang J et al. (2000) Endocrine 12, 243-247).When all of these results are considered as a group, it is clear thatdifferent forms of the ERα are possible in cells, and it is reasonableto conclude that an alternate form of ERα, possibly formed by alternatesplicing, or possibly arising from an as yet unrecognized gene, mayregulate estrogen dependent/responsive tumor cell growth.

Characterization of ERβ.

More recently, another estrogen receptor has been cloned and cDNAsequenced from rat prostate and ovary (Kuiper G G et al. (1996) ProcNatl Acad Sci USA 93, 5925-5930). It has now also been cloned from mouse(Tremblay G B et al. (1997) Mol Endocinol 11, 353-365) and human(Mosselman S et al. (1996) FEBS Lett 392, 49-53). This new receptor hasbeen named estrogen receptor beta (ERβ). Evidence that ERβ is separatefrom ERα comes from the fact that the genes are located on differentchromosomes (Enmark E et al. (1997) 82, 4258-4265). Therefore, ERβ isnot simply an alternate splicing product of the ERα gene. Furthermore,ERβ is distinguishable from ERα based on critical differences in theamino acid sequences of functional domains (Kuiper G G et al. (1996)Proc Natl Acad Sci USA 93, 5925-5930; Enmark E et al. (1997) 82,4258-4265; Dickson R B and Stancel G M (2000) J Natl Cancer Inst MonogrNo. 27, 135-145). For example, the sequence homology between the tworeceptors is 97% in the DNA binding domain, but 59% in the C-terminalligand-binding (i.e. steroid hormone-binding) domain, and only 17% inthe N-terminal domain. The ERβ N-terminal domain is much abbreviatedcompared to the ERα (Enmark E et al. (1997) 82, 4258-4265). Rat ERβcontains an 18 amino acid insert in the domain binding the ligand.Despite the significant differences in structure, ERα and ERβ bind E₂with the same affinity (Kuiper G G et al. (1996) Proc Natl Acad Sci USA93, 5925-5930; Dickson R B and Stancel G M (2000) J Natl Cancer InstMonogr No. 27, 135-145). In fact, others (Tremblay G B et al. (1997) MolEndocrinol 11, 353-365) have stated that ERβ has a slightly loweraffinity for E₂ than ERα (Tremblay G B et al. (1997) Mol Endocrinol 11,353-365). Therefore, it is important to note that if either of thesereceptors mediates estrogen-induced growth, the steroid hormoneconcentrations required for one-half maximum growth (i.e. ED₅₀), or foroptimum growth (i.e. ED₁₀₀), are expected to be about the same. Theissue of estrogen concentrations for growth required for ED₅₀ versusthose required for one-half maximum saturation of the receptors (i.e.the dissociation constant K_(d)) will be further discussed in Examplesthat follow.

ERα and ERβ Interrelationships.

Some investigators have suggested that ERα and ERβ are functionallyinterrelated (Kuiper G G et al. (1998) Endocrinology 139, 4252-4263) andthat one role of ERβ is to modulate the transcriptional activity of ERα(Hall J M and McDonnell D P (1999) Endocrinology 140, 5566-5578).Clearly however, there are significant functional differences betweenERα and ERβ. These have discussed (Gustafsson J-Å (1999) J Endocrinol163, 379-383). Also, there are functional differences expected becauseof the different pattern of steroid hormone binding shown by ERβ (KuiperG G et al. (1996) Proc Natl Acad Sci USA 93, 5925-5930). For example,ERβ binds androgens whereas ERα does not. This fact, plus the locationof ERβ in prostate indicates a new function that may be androgenrelated.

Estrogen Related Orphan Receptors.

There is also another dimension of the estrogen receptor literature thatdeserves special comment. There have been “estrogen related receptors”(ERR 1 and 2) or “orphan” receptors identified that share propertieswith ERα but do not have a known function and do not have a known ligand(Giguere V et al. (1988) Nature (Lond) 331, 91-94; Gustafsson J-Å (1999)J Endocrinol 163, 379-383). Whatever mechanism is proposed for theaction of the steroid hormone (i.e. on growth), it can be seen from thedata presented herein, and subsequently reported elsewhere (Sirbasku D Aand Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446), ittakes a significant period to reverse the effects of the inhibitor. Thisprocess cannot be simply due to a rapid effect on transcription causedby steroid hormones (e.g. via a known estrogen receptor). Cellularmetabolic events, including the transformation of E₂ to an activesteroid metabolite, may provide the growth regulating ligand for one ofthe “orphan” estrogen receptors. An alternative possibility is that thereceptor may be activated by metabolites formed from cholesterolmetabolism (Gustafsson J-Å (1999) Science (Wash DC) 284, 1285-1286). Infact, today, there are more than 70 “orphan” receptors seeking ligandsand functions (Gustafsson J-A (1999) Science (Wash DC) 284, 1285-1286).

Comparison of the Labeled E₂ Binding Dissociation Constants (K_(d)) ofSeveral Estrogen Sensitive Cell Types.

Clearly, the assays with extracts measured the same affinity bindingsites as analyses with whole cells. This offers reasonable evidence thatthe standard binding technology employed in these studies is measuringthe most common form of receptor present in cells, no matter whetherwhole cells are assayed or cell extracts. The affinity of the MTW9/PL2estrogen receptor is that which is characteristic of the ERα. The K_(d)of the receptor measures the concentration of ligand that one-halfsaturates the sites. In TABLE 10, the K_(d) values for labeled E₂ arepresented as reported and presumably represent the ERα. Only when themeasurements are specific for the β form is the designation (ERβ)included.

TABLE 10 Comparison of E₂ Binding Affinities Expressed as DissociationConstants (K_(d)) WHOLE CELLS CELL EXTRACTS CELL LINES K_(d) for E₂K_(d) for E₂ REFERENCES MTW9/PL2 2.78 × 10⁻⁹ M 1.89 × 10⁻⁹ MMoreno-Cuevas JE and Sirbasku DA (2000) In Vitro Cell Dev Biol 36,410-427 MCF-7 0.58 × 10⁻⁹ M 1.77 × 10⁻⁹ M MacIndoe JH et al. (1982)Steroids 39, 247-258 MCF-7-Mason 4.0 × 10⁻⁹ M Horwitz KB et al. (1978)Cancer Res 38, 2434-2437 Unfilled nuclear MCF-7-Mason × 10⁻⁹ M HorwitzKB et al. (1978) Cancer Res 38, 2434-2437 Filled nuclear MCF-7 × 10⁻⁹ MReddel RR et al. (1985) Cancer Res 45, 1525-1531 MCF-7-L 0.08 × 10⁻⁹ MMCF-7-M 0.07 × 10⁻⁹ M T47D × 10⁻⁹ M Horwitz KB et al. (1978) Cancer Res38, 2434-2437 Unfilled nuclear T47D 4.0 × 10⁻⁹ M Horwitz KB et al.(1978) Cancer Res 38, 2434-2437 Filled nuclear T47D 0.11 × 10⁻⁹ M ReddelRR et al. (1985) Cancer Res 45, 1525-1531 ZR-75-1 0.09 × 10⁻⁹ M ReddelRR et al. (1985) Cancer Res 45, 1525-1531 ZR-75-1 1.3 × 10⁻⁹ M Engel LWet al. (1978) Cancer Res 38, 3352-3364 H301  1.0 × 10⁻⁹ M Liehr JG andSirbasku DA (1985) In: Tissue Culture of Epithelial Cells, Taub M, ed,Plenum, New York, pp 205-234 H301 0.87 × 10⁻⁹ M Soto AM et al. (1988)Cancer Res 48, 3676-3680 GH₃ 0.25 × 10⁻⁹ M Moo JB et al (1982) In:Growth of Cells in Hormonally Defined Media, Vol. 9, Cold Spring Harbor,New York, pp 429-444 GH₃ 0.31 × 10⁻⁹ M Haug E et al. (1978) Mol CellEndocrinol 12, 81-95 Prostate and × 10⁻⁹ M (ERα) Tremblay GB et al.(1997) Mol Endocrinol 11, 353-365 Ovary 0.5 × 10⁻⁹ M (ERβ) Transfection0.05 to 0.1 × 10⁻⁹ M Kuiper GC et al. (1998) Endocrinology 139,4252-42-63 Studies (ERβ only)TABLE 10 presents only a fraction of the estrogen binding data availablein the literature. However, the K_(d) values presented arerepresentative and do show a discernable pattern. The lowest K_(d)identified in a literature search was in the range 5×10⁻¹⁰ M to1.0×10⁻¹⁰ M for the ERβ and 7×10⁻¹¹ M to 1.1×10⁻¹⁰ M for the ERα. Ingeneral, the binding affinities as estimated by K_(d) are lower forreceptors from human cells than those from rodent lines. It is importantto note that the results presented in TABLE 10 indicate that the lowerlimit of measuring estrogen receptor affinities most likely has beenreached. The use of the highest specific activity tritium labeledsteroids has been optimized and simply cannot be used to measure 10 to100-fold lower K_(d) concentrations. This opens the possibility of an asyet unrecognized ER that mediates growth effects at lower concentrationsof estrogen than either the ERα or the ERβ.

Discussion of Example 19

Evidence is provided herein that all of the ER⁺ cell lines analyzed inthis presentation show estrogenic effects (i.e. positive growthresponses significant to p<0.05 or P<0.01) obtained at 10 to more than1000-fold lower E₂ concentrations than expected from the measurement ofK_(d), with these and related cell lines. It is proposed herein thatestrogen promoted growth is mediated by an as yet to be characterizedestrogen receptor designated ERγ. In accordance with this proposal, theligand that activates ERγ may be E₂ or another cellular componentinduced, changed or modified by the action of estrogen. For example, theligand may be a lipophilic compound such as one of the intermediates ofthe cholesterol biosynthetic pathway or the phospholipid biosyntheticpathways. There is a relationship between the ERα and the ERγ, as cellsthat are growth responsive to estrogens express the ERα. This suggeststhat ERα has a functional, regulatory, gene, expression, or other typesof control relationship to ERγ in growth activated target tissues.Accordingly, ERγ may be the most accurate estimation of breast and othercancer cell growth sensitivity to estrogens, and its measurement couldserve as a valuable adjunct or replacement for the current protocolsrelying on the measurement of ERα in breast cancer.

The ERγ is also suitable for application as a diagnostic and prognostictool for other cancers such as: those of the female urogenital tractincluding ovary, uterus cervix and vagina, as well as bladder, kidney,liver, melanoma, Hodgkin's disease, pituitary adenomas and other targettissues.

Example 20 Effect of Tamoxifen Antiestrogen in Serum-Free Defined Medium

In this Example the use of one of the present cell growth assays wasused to evaluate the effects of this classical antiestrogen with “mixed”functions. A new type of growth inhibiting function for tamoxifen isidentified.

Background of Tamoxifen Effects and Clinical Applications.

The antiestrogenic effects of tamoxifen are well documented. Mostevidence suggests this compound and its active metabolite4-hydroxyl-tamoxifen prevent growth of ERα positive cells viainteraction with the receptor (Coezy E et al. (1982) Cancer Res 42,317-323; Bardon S et al. (1984) Mol Cell Endocrinol 35, 89-96; Reddel RR et al. (1985) Cancer Res 45, 1525-1531). However, it has also beensuggested that tamoxifen blocks growth factor promoted MCF-7 breastcancer cell growth (Vignon F et al. (1987) Biochem Biophys Res Commun146, 1502-1508). Also, tamoxifen has high affinity binding sites andactions distinct from the estrogen receptor (Sutherland R L et al.(1980) Nature (Lond) 288, 273-275; Phaneuf S et al. (1995) J ReprodFeral 103, 121-126). Despite its complex actions, tamoxifen haswidespread use as a treatment for breast cancer (Fisher B et al. (1998)J Natl Cancer Inst 90, 1371-1388; Jaiyesimi I A et al (1995) J ClinOncol 13, 513-529; Clinical Trial Report (1997) J Clin Oncol 15,1385-1394; Clinical Trial Report (1987) Lancet 2(8552), 171-175; ForrestA P et al. (1996) Lancet 348(9029), 708-713; Tormey D C et al. (1996) JNatl Cancer Inst 88, 1828-1833; Gundersen S et al. (1995) Breast CancerRes Treat 36, 49-53; Gelber R D et al. (1996) Lancet 347(9008),1066-1071; Raabe N K et al. (1997) Acta Oncol 36, 2550260).

Serum-Free Medium Effects of Tamoxifen.

In the present series of tests, the effects of tamoxifen (TAM) werereexamined under completely serum-free defined conditions. It isimportant to note that throughout the Examples herein, data is presentedshowing that estrogens alone have either had no effect on growth indefined medium or at most a 1.0 CPD effect that was related tosaturation density. This was true no matter if phenol red was present orabsent from the medium, as shown herein and also reported (Moreno-CuevasJ E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 447-464). Insimilar assays, 1.0×10⁻⁷ M tamoxifen was completely inhibitory with T47Dcells in culture, as shown in FIG. 107. The study shown in FIG. 107examined the concentrations of tamoxifen needed to fully inhibit T47Dcell growth in the preferred formulation of DDM-2MF serum-free definedmedium without any source of estrogens. Even phenol red was eliminated:The expected outcome was no tamoxifen inhibition. As shown, estrogenalone had only a 1.0 CPD effect in serum-free defined medium. However,tamoxifen had unexpected effects revealed by the use of serum-freedefined medium. Tamoxifen effectively arrested growth at 1.0×10⁻⁷ M.Higher concentrations were cytotoxic. It was observed in these assaysthat tamoxifen had the same effect as immunoglobulins IgA and IgM. Todemonstrate this fact another way, the experimental results presented inFIG. 108 show that estrogens completely reversed the effect of 1.0×10⁻⁷M tamoxifen. This sequence of experiments showed the same results asthose shown above with plasma IgA and IgM and ER⁺ cell lines.

Discussion of Example 20

The observation of inhibition of cell growth by a classical antiestrogendemonstrates the utility of this technology to search for otherantiestrogenic compounds. Furthermore, because of the current intensefocus on the search for SERMs (i.e. Selective Estrogen ReceptorModulators) the serum-free technology disclosed herein has particularlyuseful applications. Specific types of SERMS can be sought for differentcell types. Those SERMs that do not cause breast cancer cell growth canbe readily identified by this technology. Those SERMs with multipleactivities can be identified before conducting expensive animal testing.

The technology presented permits a clear definition of antiestrogenswith “mixed” functions (e.g. tamoxifen-like, that act at several sites)versus those with a “pure” function mediated only by the estrogenreceptor. To date, no similar easily applied in vitro method based onserum-free defined medium and secretory immunoglobulins is availablethat produces growth as an endpoint of the assay.

An entirely new function is proposed for the well-known drug tamoxifen,in which tamoxifen mimics the immune system effects on ER⁺ cancers,thereby inhibiting growth. It is believed that estrogen reverses theseeffects, not as a consequence of interaction with the classical ERα, butas a consequence of the ERγ. This mechanism is closely parallel to thatobserved with IgA/IgM and E₂, disclosed herein. Prior to the presentinvention, tamoxifen has never been linked to growth regulatory changesin the secretory immune system, nor has there been any suggestion thattamoxifen in any way mimics the inhibitory action of IgA/IgM on mucosalcells. Accordingly, certain embodiments of the present invention offernew uses for tamoxifen based on diagnostic testing to identify humanbreast, prostate, colon and other mucosal cancers that are poly-Igreceptor/secretory component positive. For example, such identificationcould be determined by immunohistochemistry or radioimmunoassay or othersuitable tests that have clinical applicability. Those tissuesdetermined to be poly-Ig receptor/secretory component positive are thencandidates for tamoxifen treatment either alone or in conjunction withother treatment modalities. The new, preferred applications of tamoxifendescribed herein is not based on the classical ERα, which has differentcriteria for its use and different tissues as potential targets.

The serum-free assay methodology described herein will be directlyapplicable to a search for tamoxifen derivative compounds showing more“immune-like” activity or other compounds with a similar activity. Theassay method is unique because of the discovery of the estrogenreversible effects of IgA and IgM and because of the results showingthat tamoxifen inhibits in the complete absence of estrogens and isreversed by natural estrogen just as happens with IgA/IgM.

The results presented show that tamoxifen inhibits the mitogenic actionof a variety of growth factors and nutrients in completely serum-freedefined culture. This effect shows the same type of “master switch”action as demonstrated by immunoglobulins, and has mechanisticimplications. The immunoglobulins shut off all growth, as did tamoxifenin these studies. As is discussed further hereinbelow, the receptormediating the effects of the immunoglobulins must possess the propertyof a “master switch” to shut down all but steroid hormone responsivegrowth. Notably, both the immunoglobulins and tamoxifen have this effecteven when a large number of “mitogens” are present. Others have reportedthat tamoxifen inhibits growth factor dependent growth (Vignon F (1987)Biochem Biophys Res Commun 146, 1502-1508), but only concluded thattamoxifen was not a “pure” estrogen. An entirely new site of action fortamoxifen is arrived at in the present disclosure.

Tamoxifen may also be an antagonist of ERγ, and may be useful in thatcapacity. The assay methods presented herein can be used to distinguishthose antiestrogens that act only on the growth estrogen receptor fromthose acting elsewhere as well. The serum-free defined medium technologypresented herein has direct application to the assay of a great varietyof drugs now in use by women either before the onset of breast cancer orafter the onset. Drugs or preparations such as antidepressants, herbalextracts, soy products, other food, plant or microorganism extracts,estrogenic creams and cosmetic preparations can be assessed forantiestrogenic or estrogenic activity. These methodologies are alsoapplicable to exploration of additional anti-androgenic compounds.Furthermore, in view of the possible role of estrogens as well asandrogens in prostate growth, this technology can be used to search forcompounds with both activities.

Example 21 Effect of Long-Term Exposure of Breast Cancer Cells to IgMUnder Serum-Free Defined Conditions

IgM Cytotoxicity after Long-Term Exposure—MTW9/PL2 Cells.

In the above examples, IgM has been demonstrated to be an estrogenreversible inhibitor of ER⁺ rodent tumor and human cancer cell growth.During these studies, visual inspection of the cultures indicated thatexperiments carried beyond 7 days with the MTW9/PL2 cells showed amarked deterioration of morphology. This suggested that exposure of theMTW9/PL2 cells to IgM might in fact be causing cell death. Suchobservations wee immediately recognized as having potential therapeuticapplications. To examine this further, MTW9/PL2 cells were incubated inserum-free defined medium DDM-2A for up to 10 days in the presence of 40μg/mL horse IgM (prepared by Custom Monoclonals International). On days0, 2, 4, 6, 8, and 10 after seeding, 10 nM E₂ was added to cultures andgrowth effects measured as cell number increases (FIG. 109). Theseresults, presented as cell numbers versus days, show that addition of E₂on or after day 8 no longer had an estrogenic effect. Conversion of thedata in FIG. 109 to CPD estrogenic effects showed very clearly that E₂addition after eight days no longer caused reversal of the IgM (FIG.110). The CPD after eight days with IgM were no different than thecontrols held in the absence of E₂ throughout the study. Clearly, IgMwas cytotoxic in eight days with MTW9/PL2 rat mammary tumor cells.

IgM Cytotoxicity after Long-Term Exposure—Human Cancer Cells.

Similar studies were done with the T471) and MCF-7A human breast cancercells in serum-free defined medium DDM-2MF. Two examples are presentedin FIG. 111 and FIG. 112, respectively. In the presence of 40 μg/mLhuman pIgM, the T47D cells and the MCF-7A cells no longer responded to10 nm E₂ by day 13. Control studies indicated the killing was IgMmediated. The conclusion was clear. IgM was cytotoxic to human breastcancer cells within two weeks. In a partial replica study with LNCaPcells (results not shown), human pIgA exposure for 14 days caused celldeath as IgM had done with T47D cells. These results have importanttherapeutic implications.

Discussion of Example 21

The results presented are the first evidence that exposure of breast andprostate cancer cells to IgA and IgM for periods of two weeks or longercan cause growth inhibition leading to cell death. At present, it is notknown if this represents some form of cytotoxicity or is due to anatural process such as apoptosis. Certainly apoptosis and cancertherapy is a dynamic current research theme, however there are noapparent previous reports in the literature related to IgA and IgMaction on mucosal cell growth and apoptosis.

A dilemma has existed for many years regarding the frequency ofmetastasis in breast, prostate and other epithelial cancers. It wouldseem that the malignant cells should populate many more new sites muchmore rapidly than actually happens in patients. To be sure, metastasesoccur at many sites, and do occur simultaneously or nearly so. However,IgA and IgM in the plasma may act to suppress the number of disseminatedcancer cells. An implication of the results of the presentinvestigations is that cancer cells in the general circulation areexposed to the effects of IgA and IgM and therefore remain inhibited orare in fact killed. Only after they are located in relativelyinaccessible sites do they not feel the full effects of IgA and IgM, andtherefore proliferate more rapidly. One example of this is the very wellknown propensity of prostate cancers to locate in bone. This is alsotrue of breast, to a significant extent. Metastatic breast and prostatecancers are very often autonomous, consistent with the presentexperimental results. Autonomous cancer cells are not inhibited by IgAand IgM, and are, therefore, free to move in plasma and proliferate atnew sites without negative immune surveillance.

Notably, the most well known human breast cancer cell line, MCF-7, wasobtained from a pleural effusion of a patient with an estrogenresponsive cancer (Soule H D et al (1973) J Natl Cancer Inst 51,1409-1416). Indeed, many researchers have sought breast cancer celllines from this fluid. The question of why this estrogen responsive andhighly immunoglobulin sensitive line survived at this new site becomesclearer, in light of the present disclosure, when it is recognized thatplural fluid is not rich in plasma immunoglobulins. Pleural fluid is afiltrate of plasma. Elevation of plasma IgA and IgM levels may havepreventative value with regard to metastasis, and therapeutic value withrespect to those tumors that are accessible to the plasmaimmunoglobulins.

Example 22 The Role of the Poly-Ig Receptor in Hormone Responsive andAutonomous Breast and Prostate Cell Growth Regulation

In this Example it was shown that the poly-Ig receptor or a poly-Ig likereceptor mediates the inhibition of cell growth by IgA/IgM. Methods ofidentifying genetic or expression defects in that receptor, andscreening methods for assessing susceptibility, and for establishing adiagnosis or prognosis in mucosal cancers are described.

Structural Properties of the Poly-Ig Receptor.

The negative response to IgA and IgM is mediated by the mucosal poly-Igreceptor or a very similar structure with the same immunoglobulinsspecificity as well as the same immunological and M_(r) properties. Thepoly-Ig receptor is a M_(r) 100,000 transmembrane protein with severalproperties that place it in the Ig superfamily of receptors (Kraj{hacekover (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315; Williams A F andBarclay A N (1988) Annu Rev Immunol 6, 381-405). The poly-Ig receptorand the secretory component from human has been cDNA cloned and DNAsequenced (Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22,2309-2315; Kraj{hacek over (c)}i P et al. (1995) Adv Exp Med Biol 371A,617-623; Kraj{hacek over (c)}i P et al. (1991) Hum Genet. 87, 642-648;Kraj{hacek over (c)}i P et al. (1989) Biochem Biophys Res Commun 237,9-20) as has the poly-Ig receptor from mouse (Kushiro A and Sato T(1997) Gene 204, 277-282; Piskurich J F et al. (1995) and bovine tissue(Verbeet M P et al. (1995) Gene 164, 329-333). Altogether, the humanpoly-Ig receptor coding sequence encompassed 11 exons. The extracellularfive domains originate from exons 3 (D1), exon 4 (D2) exon 5 (D3 andD4), exon 6 (D5), exon 7 (the conserved cleavage site to form thesecretory component), exon 8 (the membrane spanning domain), exon 9 (aserine residue required for transcytosis), exon 9 (sequence to avoiddegradation), exon 10, no known function) and exon 11 (sequence containsa threonine residue and the COOH terminus) (Kraj{hacek over (c)}i P etal. (1992) Eur J Immunol 22, 2309-2315).

With the exception of domains 3 and 4 (both from one exon), the receptorstructure follows the rule of one domain/one exon. The poly-Ig receptorbinds IgA and IgM via their Fc domains, and more particularly, via aspecific amino acid sequence (15→37) of domain 1 (Bakos M-A et al.(1991) J Immunol 147, 3419-3426). Of the other extracellular domains,only D5 is known for a specific function. It contains the disulfidebonds that covalently exchange with dimeric/polymeric IgA to form sIgAduring transcytosis. The role of this receptor in transcytosis ofIgA/IgM has been well studied with mucosal tissues and epithelial cellsin culture (Vaerman J P et al. (1998) Eur J Immunol 28, 171-182; Fahey JV et al. (1998) Immunol Invest 27, 167-180; Brandtzaeg P (1997) J ReprodImmunol 36, 23-50; Loman S et al. (1997) Am J Physiol 272, L951-L958;Mostov K E et al. (1995) Cold Spring Harbor Symp Quant Biol 60; 775-781;Schaerer E et al. (1990) J Cell Biol 110, 987-998). One serine residueis particularly important for transcytosis (Hirt R P et al. (1993) Cell74, 245-255).

Lines of Evidence Supporting Poly-Ig Receptor or a Poly-Ig Like Receptorin Negative Growth Regulation.

A series of studies and observations disclosed herein indicate that theIgA/IgM inhibition mediating receptor has the properties of the poly-Igreceptor or another receptor (“poly-Ig like receptor”) with propertiesvery similar to those of the poly-Ig receptor. From those studies, thefollowing supporting facts were gained: (1) The source of the active IgAis not the deciding factor. Plasma or myeloma derived IgA are equallyeffective. Also, species makes little or no apparent difference inactivity. IgA isolated from various species has major sequence homologyin the α heavy chains but differences in the variable chains. This isconsistent with mediation by an Fc superfamily receptor. The poly-Igreceptor is a member of this Fc binding family. (2) IgA obtained fromcommercial myeloma cell sources (especially from Zymed) containspredominantly dimeric and polymeric immunoglobulin. It is highly active.This is consistent with mediation by the poly-Ig receptor because itbinds only dimeric/polymeric IgA. (3) Cultures containing the activeCA-PS-pool II material are ≧90% dimeric/polymeric forms ofimmunoglobulins. Experiments described herein demonstrated clearly thatthis material is as active as any commercially prepared IgA in bothserum-supplemented and serum-free defined medium. This is consistentwith the expected binding of IgA to the poly-Ig receptor. (4) IgM is atleast as active, or two to three times as active as dimeric IgA, on amolar basis. Dimeric IgA is a 350 kDa complex. IgM is a 950 kDapentamer. These masses favor IgM by two to three-fold on a molar basis.Also, IgM has five Fc domains for binding, and dimeric IgA two Fcdomains. The source of the IgM can be from plasma or myeloma cells. Theyare equally effective. This is expected of the poly-Ig receptor. (5)Secretory IgA is invariable inactive as an inhibitor. It has the fiveextracellular domains of poly-Ig receptor attached. Plasma derived IgAis in contrast fully active (see FIG. 79 for IgA structures). To provethat pIgA does not have the secretory component whereas sIgA containsthe 801 kDa receptor fragment, the Western analysis in FIG. 113 wasperformed. Secretory IgA shows an 80 kDa cross-reaction band withanti-secretory component whereas pIgA shows no reaction. This was theexpected result and provides solid support for the view that the poly-Igreceptor is the mediator. Because secretory component is isolated frommilk sIgA, these results show that the secretory component used forimmunization of the rabbits was free of the other subunits in IgA. Thiswas a meaningful control for the next experiments.

In the next experiments, anti-human secretory component antiserum wasused to block the inhibiting effects of IgA and IgM. FIG. 114 shows theresults with the T47D cells in serum-free defined medium DDM-2MF withhuman plasma IgM alone and with a series of dilutions of the antiserum.As shown, 10 nM E₂ completely reversed the IgM inhibition. Dilutions of1:500 to 1:5000 also blocked the inhibition. In the insert in FIG. 114,it is shown that a control study with pre-immune rabbit serumdemonstrated that it had no inhibitor blocking activity. A similar studywas done with LNCaP cells in serum-free defined CAPM with human pIgA(FIG. 115). As shown, 10 nM E₂ completely reversed the pIgA inhibition.Anti-serum dilutions of 1:00 and 1:1000 also reversed the inhibition.Differences between the effective dilutions with T47D and LNCaP cellswas due to changes in lots of commercially prepared antiserum.Anti-secretory component antibodies completely blocked the inhibitoryeffects of IgA and IgM. These studies not only indicate poly-Ig receptormediation, but they support the view that IgA and IgM act via the samereceptor. The poly-Ig receptor is known to conduct transcytosis of bothof these immunoglobulins with very high efficiency.

To determine if IgA/IgM responsive cells expressed 1001 kDa poly-Igreceptor, the Western analysis shown in FIG. 116 was done. Amounts ofextracts of the designated cell types were analyzed with a 1:1000dilution of rabbit anti-human secretory component. As expected MDCKcells were positive. This cell line has been studied for several yearsas a model of poly-Ig receptor sorting and function. LNCaP cells showedthe same receptor (FIG. 116). Cell lines that were negative wereALVA-41, DU145, human fibroblasts, and PC3 cells (FIG. 116). As shown inmultiple experiments described herein, LNCaP cells are IgA/IgMinhibited. The results of the Western analyses show that they expressthe poly-Ig receptor.

In the final experiments of this series, pIgA was tested with two of thecell lines that were poly-Ig receptor negative by the Western analysisshown in FIG. 116. The results with DU145 cells are shown in FIG. 117.Plasma IgA was not an inhibitor. A similar study with PC3 cells is shownin FIG. 118. Again, pIgA was not an inhibitor even at 50 μg/mL. Theseresults demonstrate cells that lack the poly-Ig receptor are alsoinsensitive to pIgA.

The HT-29 colon cancer cells are known to express only the authenticform of the poly-Ig receptor. They are also negatively growth regulatedby IgM (FIG. 103). This implies that the poly-Ig receptor has morefunction than transcytosis only. This is very strong evidence in favorof the authentic poly-Ig receptor having a heretofore unrecognizedgrowth regulating function in early stage cancers of colon. The HT-29colon cancer cells are the source of a cDNA sequence for the poly-Igreceptor deposited in GENBANK. This sequence, hereby incorporated hereinby reference, is very often referred to in published reports and shownto be equal to the exons identified from normal human leukocytes thatwere the source of the genomic sequence of the poly-Ig receptor. Takentogether, all of the available data indicate that the authentic poly-Igreceptor has a new function, as identified and described herein.

Discussion of Example 22

For the first time, a relationship between immunoglobulin growthregulation and the poly-Ig receptor is demonstrated. This receptor hasin the past been studied from the perspective of a transcytosisreceptor; however, a new function for this receptor is now described.Gene changes in the authentic poly-Ig receptor gene may include pointmutations, deletions, insertions, and premature termination. Thereceptor mediating the effects of IgA/IgM may be a form arising fromalternate splicing of the original transcytosis receptor. Changes in theregulation of expression may influence the presence or absence of thisreceptor. Changes in allelic balance may affect the expression of thisreceptor and hence its function in normal, early stage cancers and inautonomous cancers. The positive correlation between the presence of ERand AR and expression of the poly-Ig receptor indicates regulation orpositive influence by steroid hormones. Without wishing to be bound by aparticular theory, it is suggested that this regulation may be at thegene expression level or at another down-stream processing point. Theactual mechanism has not yet been identified.

One of the primary themes of cancer research has been that loss of“tumor suppressor genes” causes the release of cells from negativeregulation and thereby contributes to the progression to cancer. Theevidence disclosed herein indicates that the poly-Ig receptor has a“tumor suppressor” function. It is present in cells that are regulatedby IgA/IgM and absent in cells that are insensitive to immuneinhibitors. This is a new aspect of cancer immunology that had not beenrecognized before the present invention.

For the first time, the poly-Ig receptor is connected to the D1S58linked locus that is a “hot spot” for genetic changes in breast cancer.This disclosure proposes that this locus or near neighbors contain thegrowth regulating form of authentic transcytosis poly-Ig receptor or avery similar immunoglobulin superfamily receptor. Alternately, the1q31-q41 region of chromosome 1 contains several other genes ofimmunological interest that might include the poly-Ig receptor oranother related receptor mediating the effects of IgA/IgM.

These genes are applicable for use as screens for breast and othermucosal cell cancers. They are expected to indicate susceptibility andto be used in prognosis and other diagnostic applications with humantissue and cancer samples. Analyses of allelic imbalances in thereceptor gene are also foreseen as a new tool to determinesusceptibility and prognosis for development of breast and other mucosalcancers, as will be the detection of mutations in the growth regulatingintracellular domains of the receptor. The known amino acid sequence ofthe poly-Ig receptor does not contain the immunoreceptor tyrosine-basedinhibitory motif (ITIM) common to a new family of inhibitory motifreceptors (Cambier J C (1997) Proc Natl Acad Sci USA 94, 5993-5995).Other amino acid sequences may serve this same function.

Example 23 IgG1 and IgG2 as Immunoglobulin Regulators of Estrogen andAndrogen Responsive Cancer Cell Growth

A role for IgG1 and IgG2 as immunoglobulin regulators of estrogen andandrogen responsive cancer cell growth is described in this Example,together with methods describing how to use those IgG subclasses toidentify the Fcγ receptor that mediates their inhibitory effect. Use ofthe receptor, and its gene for assessing susceptibility to cancer, andin diagnostic, gene screening and other applications is also addressed.

Background Regarding IgG Subclasses.

The major immunoglobulins secreted as mucosal immune protectors includeIgA, IgM and IgG. In human serum, the percent content of IgG, IgA andIgM are 80, 6 and 13%, respectively. In humans, the major subclasses ofIgG are IgG1, IgG2, IgG3 and IgG4. These are 66, 23, 7 and 4% of thetotal IgG, respectively. The relative content of human immunoglobulinclasses/subclasses in adult serum follow the orderIgG1>IgG2>IgA1>IgM>IgG3>IgA2>IgD>IgE (Spiegelberg H L (1974) Adv Immunol19, 259-294). When the serum concentrations of immunoglobulins arecompared to those in exocrine secretion fluids, the relative contentschange dramatically (Brandtzaeg P (1983) Ann NY Acad Sci 409, 353-382;Brandtzaeg P (1985) Scand J Immunol 22, 111-146). For example incolostrum (a breast fluid secretion), secretory IgA is Z 80% of thetotal immunoglobulins. IgM is ≦10% of the total. IgG represents a fewpercent. In human colostrum and milk, IgG1 and IgG2 are the majorsubclasses of IgG (Kim K et al. (1992) Acta Paediatr 81, 113-118).Clearly, comparison of serum and mucosal fluid concentrations indicateselective immunoglobulin secretion. The secretion mechanism for IgA andIgM are well described. Conversely, there is a fundamental questionsurrounding IgG secretion. There is no “J” chain present in IgG1 andIgG2. From the known facts of transcytosis/secretion of immunoglobulins(Johansen F E et al. (2000) Scand J Immunol 52, 240-248), it is unlikelythat IgG secretion is mediated by the poly-Ig receptor. An epithelialreceptor specific for IgG1 has been reported in bovine mammary gland(Kemler R et al. (1975) Eur J Immunol 5, 603-608). Apparently, itpreferentially transports this class of immunoglobulins from serum intocolostrum. Despite this 1975 report however, the receptor has not beenchemically or structurally identified nor has the mechanism of transportof IgG monomers been satisfactorily defined. Certainly no growthfunction was ascribed to this “IgG1 receptor” in the 1975 Kemler et al.report. It is possible that this receptor is a member of a large groupnow designated as Fc receptors (Fridman W H (1991) FASEB J 5,2684-2690), but there is one study with IgG showing that, of 31different long-term human carcinoma cell lines, including breast, “alllines were found to be consistently Fc receptor negative” (Kerbel R S etal. (1997) Int J Cancer 20, 673-679). One possible candidate for theepithelial transport of IgG1 is the neonatal Fc receptor (Raghavan M andBjorkman P J (1996) Annu Rev Cell Dev Biol 12, 181-220). However, thereis no indication yet of the presence of this receptor in adult mucosaltissues.

Value of Assessing IgG Subclasses for Activity.

Although the IgG class is lowest in concentration in secretory fluids,it is still physiologically important because of its capacity toneutralize pathogens by various mechanisms. The human clinicalimportance of understanding and measuring IgG subclasses has beengrowing steadily. From a few clinical reports per year in 1970, theliterature now exceeds four hundred reports a year. These assays arevaluable for several reasons, including the following: (1) they providea clearer picture of an individual's susceptibility to disease; (2) anawareness that treatment for subclass deficiencies is important; (3) thesubclasses can be used to assess the state of a number of diseases; and(4) the IgG subclass difference between ethnic groups and differentraces is a potential area for expanded control of disease. The presentinvestigations showed that bulk purified mixtures of all subclasses ofhorse and rat IgG were not estrogen reversible inhibitors for MTW9/PL2rat mammary tumor cells. These results were further examined, asdescribed below.

Test of Rat IgG Subclasses as Estrogen Reversible Inhibitors of MTW9/PL2Rat Mammary Tumor Cell Growth.

The IgG subclasses of rat are IgG1, IgG2A, IgG2B and IgG2C. These IgGs,obtained from commercial sources previously identified herein, weretested at 15 μg/mL with MTW9/PL2 cells in DDM-2A serum-free definedmedium (FIG. 119). All four IgG subclasses were compared to rat pIgA andrat pIgM. The latter two were estrogen reversible inhibitors, asexpected (FIG. 119). However, the four IgG subclasses were notinhibitors at a concentration that was effective with IgA or IgM. Theestrogenic effects recorded in cultures with them were no larger thanseen in serum-free defined medium alone (FIG. 119). Clearly, IgG are noteffective steroid hormone modulators in rat.

Test of Human IgG Subclasses as Estrogen Reversible Inhibitors of Breastand Prostate Cancer Cell Growth.

The subclasses of human IgG are IgG1, IgG2, IgG 3 and IgG4. They areformed with both λ and κ light chains. A series of studies wasperformed, and it was found that with human breast cancer cells, onlyIgG1κ was a significant estrogen reversible inhibitor. FIG. 120 shows acomparison of its activity to human pIgA and pIgM. At 40 μg/mL, it was37% as effective as pIgM. A similar study with LNCaP cells showed thatonly IgG1κ had activity greater than the estrogenic effect seen in CAPMserum-free defined medium only (FIG. 121). However, in some experimentswith prostate cells, IgG2a also showed androgen reversible inhibitoryactivity (FIG. 122). Based on these studies, it is concluded that IgG1and IgG2 have small but measurable androgen reversible activity with AR⁺human prostate cancer cells.

Discussion of Example 23

The effect of IgG1κ raises an issue not encountered with IgA or IgM. Thepreference for the κ light chain implies that a different receptormediates the effects of this immunoglobulin. This immunoglobulin mayhave greater inhibitory effects on normal breast or prostate cells thatit has on ER⁺ and AR⁺ cancer cells. Part of thetransformation/progression process leading to hormone responsive cancersmay be an attenuation of the effectiveness of IgG1× as an inhibitor. Thepresent IgG1 observations have other applications, as well, includingthe measurement of the IgG1κ subclass in different populations such asblack American, Asian, white, Native American and Hispanic withcontrasting susceptibilities to breast and prostate cancer, orindividuals within any one ethnic group, may provide additionalinformation and confirmation of the usefulness of such measurements.These measurements can be made in bodily fluids or plasma. Measurementin milk and breast fluid may provide an indication of susceptibility tothe development of breast cancer.

Irrespective of the receptor that mediates the growth response of IgG1κor IgG2, this receptor will be a candidate for the missing transcytosisreceptor for IgG. Its molecular identification has utility in diagnosticspecimens of breast, prostate and other cancers and can be used todetermine new uses of the immune system for therapeutic applications.Once it is completely identified, the receptor that mediates theIgG1/IgG2 growth inhibition effects will provide another target fordevelopment of compounds that mimic the immune system inhibition ofcancer cell growth. As described above with respect to the gene for thepoly-Ig receptor, the gene encoding this IgG receptor will also beuseful as a locus for analysis of genetic susceptibility to breast andprostate cancers, as well as other types of mucosal and epithelialcancers of humans.

Example 24 Mediation of IgG1κ Effects by a Fc-Like Receptor

In this, example the probable mediating receptor for IgG1κ cancer cellgrowth inhibiting effects is further described and applications forusing the gene encoding this receptor as a genetic screening tool to aidin assessing genetic susceptibility are discussed.

It is Highly Unlikely that IgG1 Acts Via the Poly-Ig Receptor.

The poly-Ig receptor has a requirement for “J” chain for binding (henceits specificity for dimeric/polymeric IgA or pentameric IgM each ofwhich has one J chain). Also, as shown in TABLE 11, Fcγ receptors arelocalized in leukocyte series or bone marrow origin cells. There is noconvincing evidence in the literature of their presence in epithelialcells or in secretory cells of the mucosa. The IgG1 inhibition-mediatingreceptor sought in the present study is one analogous to the Fcγ in twosignificant properties. First, it binds monomeric IgG1 via the Fc domainof the immunoglobulin with some participation of the κ light chain.Second, the receptor has inhibitory activity akin to a new family of Fcreceptors. The amino acid sequence of the new IgG1κ receptor is expectedto have an immunoreceptor tyrosine-based inhibitory motif (ITIM)(VxYxxL) common to a new family of inhibitory motif receptors (Cambier JC (1997) Proc Natl Acad Sci USA 94, 5993-5995). Alternatively, otheramino acid sequences may serve this same function. The Fcγ family ofreceptors contains members that possess a very special property. Theyare expected to mediate growth inhibition. The methods of identificationare outlined below.

TABLE 11 Properties of the Fcγ Family of Receptors Fcγ R1 Fcγ RII FcγRIII (CD 64) (CD 32) (CD 16) IgG1 Binding K_(a) = 10⁸ M⁻¹ K_(a) = 2 ×10⁶ M⁻¹ K_(a) = 5 × 10⁵ M⁻¹ Binding Order IgG1 > IgG1 > IgG1 = IgG3 =IgG3 = IgG3 IgG4 > IgG4 > IgG2 IgG2 Found in these MacrophagesMacrophages Natural Killer Cells Cell Types Neutrophils NeutrophilsMacrophages Eosinophils Eosinophils Neutrophils Platelets Eosinophils BCellsIt should be noted that none of these receptors has previously beenidentified in mucosal cells. Identification of one of these, or a highlyrelated growth inhibitory Fc receptor, in mucosal cells will be asignificant advance with many practical and clinical applications.

Discussion of Example 24

The amino acid sequence of a new Fc family receptor may includeimmunoreceptor tyrosine-based inhibitory motif (ITIM) common to a newfamily of inhibitory motif receptors (Cambier J C (1997) Proc Natl AcadSci USA 94, 5993-5995). Fc receptors of mucosal cells that may includeone of the known members of the family of ITIMs, or may contain anotheramino acid sequence or sequences that serve this same function, are thesubject of ongoing investigation. Once the sequence is identified, thegenetic mapping to a specific chromosome number and locus is expected.The genomic DNA sequence of the new receptor (or existing receptor, ifalready known), including introns and exons, is also expected. Onceidentified, this receptor will find use as a genetic screening tool forgenetic susceptibility to breast and prostate and other mucosal cancers,in addition to, or analogous to, conventional breast and prostatescreening technologies. Additionally, the IgG1 mediating receptor willbe employed for diagnostic and clinical applications, as furtherdiscussed hereinbelow. Detection of mutations and changes associatedwith progression from normal cells to autonomous cancer cells are usingthis receptor gene is foreseen. Methods of detecting changes inregulation or expression of the receptor due to allelic imbalances inthe receptor gene are also foreseen as a new tool to determinesusceptibility and prognosis for development of breast and other mucosalcancers. Detection of other regulatory and developmental changes arealso made possible by this receptor and its gene.

Example 25 Immunoglobulin Inhibitors as Tools for Identifying theReceptors that Mediate the IgA/IgM/IgG Cell Growth Regulating Effects

This Example describes how IgA, IgM and IgG1 can serve as biologicalreagents or tools in establishing the identity of the inhibitionmediating receptors.

The Mediating Receptors—Inhibitory Function.

It has been made clear by the results presented herein, and in co-ownedconcurrently-filed U.S. patent application Ser. No. 09/852,958PCT/US2001/15183 entitled “Compositions and Methods for DemonstratingSecretory Immune System Regulation of Steroid Hormone Responsive CancerCell Growth,” hereby incorporated herein by reference, that themediating receptor for the serum-borne agent has special properties. Asdiscussed above, serum contains a great variety of mitogenic agents. Onthis point the present results in 50% (v/v) serum were especiallyrelevant. This concentration of serum is a rich source of mitogensincluding insulin and the insulin-like growth factors. Nutrients andother serum components also have growth-promoting effects. Examplesinclude diferric transferrin, unsaturated fatty acids bound to albumin,complex lipids and ethanolamine. The broad range of different “mitogens”present in defined medium are described elsewhere (Riss T Land SirbaskuD A (1987) Cancer Res 47, 3776-3782; Danielpour D et al. (1988) In VitroCell Dev Biol 24, 42-52; Ogasawara M and Sirbasku D A (1988) In VitroCell Dev Biol 24, 911-920; Karey K P and Sirbasku D A (1988) Cancer Res48, 4083-4092; Riss T L et al. (1988) In Vitro Cell Dev Biol 24,1099-1106; Riss T L et al. (1988) In Vitro Cell Dev Biol 25, 127-135;Riss T L and Sirbasku D A (1989) In Vitro Cell Dev Biol 25, 136-142;Riss T L et al. (1986) J Tissue Culture Methods 10, 133-150; Sirbasku DA et al. (1991) Mol Cell Endocrinol 77, C47-055; Sirbasku D A et al.(1991) Biochemistry 30, 295-304; Sirbasku D A et al. (1991) Biochemistry30, 7466-7477; Sato H et al. (1991) In Vitro Cell Dev Biol 27A, 599-602;Sirbasku D A et al. (1992) In Vitro Cell Dev Biol 28A, 67-71; Sato H etal. (1992) Mol Cell Endocrinol 83, 239-251; Eby J E et al. (1992) AnalBiochem 203, 317-325; Eby J E et al. (1993) J Cell Physiol 156, 588-600;Sirbasku D A and Moreno-Cuevas J E (2000) In vitro Cell Dev Biol 36,428-446). From the present results, clearly, the immunoglobulininhibitor(s) also block the growth effects of all those mitogens, andsteroid hormones are selectively capable of reversing the effects of theinhibitor(s). Plainly, as predicted by the estrocolyone hypothesis,serum contains an inhibitor that has a dominant role in the regulationof proliferation of steroid hormone target cells. These inhibitors willhave biological implications extending well beyond estrogen and androgentarget tissues. Because of its “master switch” character, the newlyidentified immunoglobulin inhibitors have many practical industrialtesting and manufacturing uses as well as many beneficial clinicalapplications.

The Receptor Mediating IgA/IgM/IgG Inhibitory Effects.

The results shown herein strongly indicate that the IgA/IgM growthinhibition is mediated either by the poly-Ig receptor or a very closelyrelated receptor. Establishing a growth regulating function for this“transcytosis” receptor will open new directions in medical diagnosis,treatment and prevention of cancers of mucosal epithelial tissues. Itwill be determined whether the poly-Ig receptor, or a poly-Ig likereceptor, mediates the growth regulating effects of IgA on human breastand prostate cancer cells in culture. For this study, the poly-Igreceptor in these cancer cells will be identified using well-known PCRcloning technology, ¹²⁵I-labeled IgA chemical cross-linking and Westernand immunohistochemistry methods that have been described in theliterature.

Next, blocking polyclonal antibodies or blocking monoclonal antibodieswill be employed to show that the poly-Ig receptor mediates the growthresponse. The antibodies will be raised against the poly-Ig receptorusing known techniques. Reversal of the inhibitory effect of IgA and IgMby blocking the poly-Ig receptor will suggest that the poly-Ig receptoris not just a simple transport receptor, but that it has a central rolein breast and prostate cancer cell growth regulation. There is noexisting paradigm for breast or prostate cell growth regulation thatinvolves the poly-Ig receptor or for that matter any receptor specificfor the IgA class of immunoglobulins including Fcα receptors (Fridman WH (1991) FASEB J 5, 2684-2690).

The different forms and domains of IgG, IgA and IgM that act asinhibitors of normal prostate and breast and other mucosal epithelialcell growth and the hormone responsive and hormone autonomous forms ofthese cancers in serum-free defined culture medium will be determinedand used as tools to evidence or confirm the identity of the receptor(s)responsible for mediating the growth regulatory effect. The propertiesof the ligand that elicits a response will be evidence supporting theidentity of the receptor. Poly-Ig receptor is activated by Fc-domains asare Fcγ receptors. Normal cells are likely to be most inhibited by IgG,IgA and IgM, whereas the ER⁺ and AR⁺ cells will likely be inhibitedprimarily by IgA/IgM, and ER⁻ and AR⁻ cells will likely not be inhibitedby any of the three classes of immunoglobulins, as predicted by theconceptual model described below. The methods employed will includedirect tests of the activity of IgG, IgA and IgM on cell growth as wellas assessment of the activity of specific size forms and Fc versus Fabfragments. Antibodies such as anti-J chain and anti-Fe will be used toextend these studies to demonstrate that the Fc is the active domain andthat Fc binding receptors are involved.

More specifically, AR⁺ LNCaP cells, the AR⁻ PC3 and DU145 cells, and theAR⁺ ALVA-41 cells will be studied. Normal human prostate and breastepithelial cells will be obtained from Clonetics. Growth assays will bedone in completely serum-free CAPM (prostate) and DDM-2MF (breast), asdescribed above. IgA1 and IgA2 will be purified from human serum andcolostrum, using techniques that are well known and have been describedin the literature. Initial small samples will be obtained from acommercial supplier such as The Binding Site (San Diego, Calif.). Themonomeric, dimeric and polymeric forms of IgA will be separated usingtechniques that are well known and have been described in theliterature. If only IgA2 has activity, it will be further separated intothe A2(m)1 and A2(m)2 allotypes, using well-known techniques that havebeen described in the literature. Because the initial IgA/IgM inhibitorpreparations evaluated in the present studies were mostly dimeric andmonomeric, those forms are expected to be the most active in the futureseries of tests. Confirmation that the most active forms aredimeric/polymeric IgA/IgM will be strong evidence for poly-Ig receptormediation. Should the monomers be revealed as the only active inhibitorforms, however, it would favor Fc or Fc superfamily receptors, in whichcase the Fcα will be investigated as a possible mediator.

IgA will be fragmented with a specific protease to yield Fc and Fabfragments from IgA, using techniques that are well known and have beendescribed in the literature. The Fab and Fc fragments of IgM will beobtained using a Pierce Chemicals kit based on immobilized trypsin. Faband Fc fragments of IgG1 will be obtained using another Pierce kit. Ifonly Fc fragments of IgA and IgM are active, mediation by the poly-Igreceptor is likely. If the Fc of IgG1 is active, it will indicate an Fcreceptor as the mediator.

The immunoglobulin inhibitors will also be used as tools or biologicalreagents to confirm whether IgG acts via a receptor different thanIgA/IgM. Based on the results reported above, identification of Fcγ likereceptors and the poly-Ig receptor (or related receptor) with normalcells, ER⁺ cells and AR⁺ cells is expected, and no functional receptorsare expected in ER⁻ cells or AR⁻ cells. ¹²⁵I-labeled IgG1, IgA and IgMwill be prepared using chloramine T or Iodogen® beads or coated tube(Pierce Chemicals kits). Binding parameters, binding constants, analysesof the effects of reciprocal additions of labeled and unlabeledimmunoglobulins to identify separate or similar binding sites, anddetermination of the effects of addition of purified secretory componenton IgA and IgM binding will be performed as previously described orusing well known published techniques. Specific binding will be as totalbinding minus binding in a 100-fold excess of unlabeled protein. Foreach form with activity, time, concentration and temperature dependenceof binding will be assessed. Scatchard analysis will be used to estimatethe number of sites per cell and the association constants (K_(a)).Reciprocal competitions with unlabeled and labeled immunoglobulins willbe used to define interaction with the same or different receptors. Thislatter point is important because binding of both IgA and IgM to thesame site strongly favors the poly-Ig receptor and plainlycontra-indicates Fcα (IgA) or Fcμ (IgM) receptors, which are members ofa superfamily in which each member is specific for a (monomer) class ofimmunoglobulins. In addition, the effects of blocking antibodies such asanti-secretory component, anti J chain and anti Fc will be assessed withall three cell types. Where indicated, chemical cross-linking with¹²⁵I-labeled Ig will be performed to define the mass of the receptors.Optionally, metabolic labeling and/or immunoprecipitation techniqueswill be used instead, employing well-known techniques that have beendescribed in the literature.

Western immunoblotting with normal, steroid hormone receptor positiveand steroid hormone receptor negative cell types will be performed toidentify the receptors present. Immunohistochemistry will be applied toidentify the poly-Ig receptor and Fcγ receptors on all three types ofcells using the blocking antibodies. Using a full-length human poly-Igreceptor cDNA clone, S1 nuclease protection assays will be conductedwith RNA from normal prostate and breast cells, ER⁺ and ER⁻ breastcancer cells, and AR⁺ and AR⁻ prostate cancer cells to identify mRNA. Inthe cases of ER⁺ and AR⁺ or ER⁻ or AR⁻ cells, this method will help toidentify truncated or otherwise altered receptors or non-functionalreceptors. As described in certain of the preceding examples, Westernblots have already been conducted, as well as cell growth assays withreceptor blocking antibodies. The remaining analyses will be done withnormal cells as well as all other ER⁻ or AR⁻ lines. All blockingantibodies are dialyzed against buffer containing charcoal to removeinterfering steroid hormones. Rabbit polyclonal anti secretory componentwill be raised (e.g., by HTI BioProducts, Ramona, Calif.) and rabbitpolyclonal anti-human J chain and specific antibodies against the Fcreceptors for IgG and IgA are commercially available (Accurate). Thespecificity of all antiserum will be checked by Western analysis.

To identify the receptors mediating the androgen reversible inhibitionof normal and/or AR+ cells, PCR cloning methods will additionally beused to determine the cDNA sequences of the poly-Ig receptor and Fcγreceptors from normal, AR⁺ and, if indicated, from AR⁻ cells. Thismethod will provide clear answers to the question of the relationship ofthe human poly-Ig receptor and Fcγ receptors to immune system negativeregulation. It is expected that the receptors will be found to be eitheridentical to known sequences or altered in sequence to convert them to“inhibitory motif” receptors. Based on the known cDNA sequence of thepoly-Ig receptor from HT-29 cells, PCR cloning technology will beapplied to obtain a full-length clone from the LNCaP and T47D cells.Ongoing investigations are directed to comparing receptor sequences fromnormal prostate and breast cells to identify any changes. Based on theknown sequence of the FcγRIIB1 receptor, these same studies will berepeated. The receptors identified by cloning will be examined for theimmunoreceptor tyrosine-based inhibitory motif (ITIM) amino acidsequence I/VxYxxL or related sequences. Concomitantly, the cells will beexamined by Western analysis for SHP-1 and SHP-2 phosphatase mediatorsof the inhibition of growth factor activity. These markers are not onlyassociated with the inhibitory motif but also other inhibitoryreceptors. More specifically, an LNCaP and T47D full-length poly-Igreceptor clone will be prepared and compared to the reported sequence ofthe poly-Ig receptor. The same technology will be applied to the poly-Igreceptor from normal prostate cells, and, if indicated, from the AR⁺lines. Because these cell lines are expected to express the knownpoly-Ig receptor, or a related form, the PCR approach is applicable. Thesame approach will be used with the Fcγ like receptor. However, in thiscase, because these receptors are predominantly lymphoid origin, theform in epithelial cells may be substantially different. Standardcloning methods will be employed to obtain the complete cDNA sequence ofthe Fcγ like receptor from normal and LNCaP cells. Total RNA will beextracted and mRNA purified by oligo dT cellulose chromatography (alsofor Northern analysis). cDNA synthesis will be done with oligo dTprimers and AMV reverse transcriptase followed by Rnase H to remove RNA.Second strand synthesis will be done with hexameric random primers andDNA pol. I. Treatment with T4 DNA pol, Rnase H and Rnase A creates bluntends. EcoR1 methylation is followed by EcoR1 linkers and ligation into acloning vector. (Stragene) vectors based on λgt10 (hybridizationscreening) and λgt11 (secretory component antibody screening). Bothvectors will accept inserts larger than the receptor. The cDNA sequenceof human poly-Ig receptor known is the genomic sequence. These will beused to prepare sequence specific primers for PCR. The primers willencompass the 5′ and 3′ non-coding sequences to ensure a complete cDNA.The PCR products will be subcloned using the TA kit from Invitrogen. Thesequencing of PCR clones will be done by the dideoxy chain terminationmethod (Lone Star Labs, Houston, Tex.). From these, determination ofwhether there have been significant alterations in the receptor duringthe transition from normal to ER⁻ and AR⁻ cancer cells is expected. Fromsequence data, the ITIM amino acid sequences indicating an inhibitorymotif receptor will be sought. It is important to note, however, thatthe absence of these sequences does not necessarily rule out aninhibitory function. The Western analyses for SHP-1 and SHP-2 will bevaluable as an indication of an inhibitory function even in the absenceof ITIM or when the ITIM is in a modified form.

Discussion of Example 25

Without wishing to be bound by a particular theory, it is proposed thatthe inhibitory effect of IgG1 is more marked with normal cells than withER⁺ or AR⁺ cancer cell lines and an early step in the pathway tomalignancy involves loss by the cell of IgG1 regulation. Frompreliminary investigations, it appears likely that the IgA and IgMreceptors are a common poly-Ig receptor (or a poly-Ig like receptor),which in normal cells is expected to be the same as in steroid hormonereceptor positive cell lines. In contrast, the IgG1 receptor, likely anFc gamma type receptor, is expected to either be either geneticallyaltered, or its expression altered by changes in other controls, toreduce the receptors in ER⁺ and AR⁺ cell lines. The demonstration thatIgG1 has a major growth inhibiting effect on normal cells may lead toimmunization against breast cancer by administering or enhancing IgG1 inat-risk tissues. Characterization of an inhibitory role for IgG1 via anFcγ-like receptor is expected to lead to important innovations inmedical diagnosis, treatment and prevention of cancers of mucusepithelial tissues.

Example 26 Conceptual Model for Cascading Loss of Immunoglobulin Controlin Progression from Normal Cells to Steroid Hormone Responsive andAutonomous Cancers

Concept.

The isolated inhibitors, now identified as IgA, IgM and IgG1, controlledbreast and prostate cell growth by acting as a steroid hormonereversible inhibitor even when tested under the very rigorous conditionsof serum-free defined culture. These active natural inhibitors arepresent in blood, bodily secretions and mucosal epithelial tissues. Theisolated inhibitors readily prevented the growth of these types ofcancer cells when they were still in the early (i.e., hormoneresponsive) stage, but not in the late, non-hormone responsive stage.These results have many implications with regard to the diagnosis,genetic screening, treatment and prevention of breast, prostate, colonand other mucosal cancers. Without wishing to be bound by a particulartheory, considering the present discoveries and experimental resultsand, a new conceptual model for understanding how estrogens cause ER⁺breast cancer cell growth and for understanding how the naturalprogression of breast cancers occurs to give rise to highly malignant(and dangerous) hormone autonomous forms is proposed. This same model isapplicable to other mucosal tissues that respond to the steroid hormonefamily of hormones, including androgens and thyroid hormones.

Progression Concept Based on the Breast Cancer Model—GenerallyApplicable to Mucosal Tissue Cancers.

It is well established that breast cancers pass through a characteristicnatural history that involves a gradual evolution from near normalgrowth patterns into cancers that are completely steroid hormoneautonomous (i.e. they are no longer stimulated by steroid hormones).These are usually designated estrogen receptor negative (ER⁻). Asdisclosed herein, it has been found that autonomous (ER⁻) breast canceris accompanied by a loss in sensitivity to IgA or IgM. Fully autonomousbreast cancers are not inhibited by these secretory immunoglobulins. Inlight of the results described herein, it appears that autonomous breastcancers lack the poly-Ig receptor that mediates the growth inhibitingeffects of IgA and IgM. These results are of special significancebecause for the first time they pinpoint a specific genetic change (i.e.in the poly-Ig receptor) that might account for the majority (i.e.approximately 75%) of breast cancers termed “sporadic” and for whichthere is as yet no clear genetic change identified. Indeed, theseresults also provide an excellent opportunity to implement gene therapybased on reintroduction of the poly-Ig or poly-Ig like receptor intocompletely autonomous cancers to regain immunological regulation.

It is well established in the literature that IgG1 is present in serumduring childhood, when breast tissue growth is precisely regulated tobody size (isometric growth). The other inhibitors, IgA and IgM, arevery low at this time, but increase in serum at puberty. Because adultwomen have increased positive stimuli for breast cell proliferation dueto estrogen production, the presence of IgA and IgM may provideadditional protection. It is now proposed that alterations in immuneregulation lead to the progression of breast and prostate cells fromnormal control to ER⁺ and AR⁺ cancer cells and that additionalalternations in immune control contribute to the development of fullyautonomous cancers, according to the following model presented in TABLE12:

TABLE 12 Model for Progression of Steroid Hormone Dependent Cancers fromNormal Growth Regulation by the Immune System to Steroid ResponsiveCancers and on to Fully Hormone Autonomous Cancers

Inhibitory Motif Receptors.

The receptors mediating the immune response regulation must be at orvery near the beginning of the onset of breast cancer. Using the toolsdeveloped in the present series of investigations, it is expected thatinhibitory motif receptors for these immunoglobulins will be identified.It is now proposed that the mediating receptors are members of the Igsuperfamily, which includes Fc receptors and a new class of Iginhibitory motif receptors. This new class of receptors has emergingimportance because of the increasing recognition of the role of negativeregulation of cell growth. These receptors have both common and uniqueproperties. They bind immunoglobulins via the Fc domains and hence canbe classified as Fc receptors. One of these is, in fact, FcγRIIB thatbinds IgG1 (TABLE 12) and causes inhibition of antigen activation of Bcells. There are many other examples (Cambier J C (1997) Proc Natl AcadSci USA 94, 5993-5995). Among these are more than 15 receptors nowdesignated Signal-Regulatory Proteins (SIRPs). These all express aspecial inhibitor motif of six amino acids (I/VxYxxL) that is nowreferred to as the “immunoreceptor tyrosine-based inhibitory motif” orITIM. One of the most marked characteristics of the ITIM containingSIRPs is that this motif recruits two phosphatases (SHP-1 and SHP-2) toresult in the inhibition of all growth factor dependent proliferation.This is similar to what was observed with IgG1, IgA and IgM and ER⁺breast cancer cells and AR⁺ prostate cancer cells serum-free definedmedium. This work is expected to aid in the identification of themissing genes for sporadic breast cancers and a more completeunderstanding of the cascade of gene changes that lead to complete lossof immune control of breast cell growth.

Similarly, it is suggested that alterations in immune regulation alsolead to the progression of prostate cells from normal control to AR⁺cancer cells and that additional alterations in immune controlcontribute to the development of AR⁻ fully autonomous cancers. Furtherstudies are directed at identifying a cascade of gene changes leading tocomplete loss of immune control of cell proliferation.

Similarly, it is also proposed that alterations in immune regulationalso lead to the progression of colon cancer cells from thyroid hormonereceptor (THR) normal control to THR⁺ cancer cells and that additionalalterations in immune control contribute to the development of THR⁻fully autonomous cancers. Further studies are directed at identifying acascade of gene changes leading to complete loss of immune control ofcell proliferation

Tests to determine whether steroid hormone independent breast andprostate cancer cell growth results from either the loss of the poly-Igreceptor or an inactivation of its function are a focus of continuinginvestigations. A series of steroid hormone dependent and steroidhormone independent breast and prostate cancer cell lines will becompared for their inhibitory growth responses to IgA, the presence ofpoly-Ig receptor m-RNA, the expression of the receptor by ¹²⁵I-IgAbinding analysis and immunohistochemistry localization of receptor.Detection of an absence of the receptor or an inability to bind IgA willsuggest that cancer cell autonomy arises due to a loss of secretoryimmune system regulation. Such a result would be entirely new in thefield of hormone dependent cancers and would provide a new immunemechanism responsible for conversion from hormone dependence toautonomy. New immunotherapies can be developed based on activating thereceptor in hormone responsive cancers and new gene therapies based onreestablishing the function of this receptor in autonomous breastcancers.

Ongoing investigation is directed at resolving whether hormoneautonomous breast cancer cell lines have functional poly-Ig receptors.The ER⁻ cell lines to be studied are the MDA-MB-231, BT-20, MDA-MB-330the non-tumorus BBL-100, and the Hs578t and Hs578Bst. Each will beevaluated for growth in serum-free medium±IgA and ±E₂. This study willdetermine if autonomous cells have lost immune system negativeregulation. To determine if the receptor is lost, the S1 nucleaseprotection assays will be used to seek its mRNA. A kit from AMBION willbe used. In addition, ¹²⁵I-I labeled IgA will be used to determinespecific binding characteristics as described above.Immunohistochemistry will be used to confirm and/or extend the bindingdata. If the receptor mRNA and protein are absent, these methods shouldconfirm that fact. Alternatively, if they are present but nonfunctional,these methods should also confirm that fact.

Discussion of Example 26

The proposed model of progression of mucosal cancers from normal cellsto fully autonomous cancers is based on the experimental resultspresented, and has not been suggested prior to the present invention. Aspreviously stated, there has also been no previous recognition of theroles of IgA, IgM and IgG1 in breast, prostate, or other mucosalcancers. The cancer progression model has diagnostic implications. Forexample, breast, prostate and other cancers can be examined for contentof the IgA, IgM and IgG1 receptors, as an indicator or aid todetermining the stage of the cancer. This information can be compared tothe determination of estrogen receptor and progesterone receptor statusto aid in decisions regarding immunotherapy with immune modulators orthe immunoglobulins or the use of combined anti-hormone and immunetherapy modalities. Tumors that are negative for all of theimmunoglobulin receptors are prime candidates for gene therapy toreplace the receptors and thereby reestablish immune surveillance, asfurther described in a subsequent example.

Example 27 Role of TGFβ in Breast Cancer Predicts the CellularProgression in Early Onset Breast Cancer

This Example describes a new model for TGFβ and secretory immune systemroles in cancer progression in early onset breast cancer. A “linear”progression model (e.g., normal breast cell→ER⁺ cancer cell→ER⁻ cancercell) has been generally accepted for many years (Furth J (1959) CancerRes 3, 241-265; Heppner G H (1984) Cancer Res 44, 2259-2265). Inconformity with the linear progression concept, a modified model ofhuman mucosal cell progression is presented (shown in TABLE 12) thatoutlines sequential passage of normal cells, to steroid hormonestimulated cancers that in turn give rise to steroid hormone autonomouscancers, and includes the proposed roles played by the immunoglobulininhibitors.

There exist, however, pronounced factual issues that are not adequatelyaddressed by the linear progression model. For example, it is known thatearly onset (i.e. pre-menopausal) breast cancers are 60 to 70% ER⁻ orsteroid autonomous. This fact is difficult to explain under a strictlylinear progression model because during this time (i.e., thepre-menopausal stage) female levels of estrogen are high, and thereforeshould favor outgrowth of estrogen responsive tumors. Considering all ofthe foregoing and a number of seemingly unrelated observations, in lightof the TGFβ experimental results obtained herein, an alternative newconcept, or model, of “progression” in early onset breast cancer hasbeen reached. This proposed model is illustrated as a schematic flowdiagram in FIG. 123. This model suggests an alternative or additionalsequence for cancer progression that does not in all cases require thetransition to ER⁺ or AR⁺. As shown previously herein, TGFβ has little ifany inhibitory effect on ER⁺ breast cancer cells (FIGS. 25 and 26).However, it is also well established that TGFβ is a very potentinhibitor of normal breast epithelial cell growth (Hosobuchi M andStampfer M R (1989) In Vitro Cell Dev Biol 25, 705-713; Daniel C W etal. (1996) J Mammary Gland Biol Neoplasia 1, 331-341). Furthermore, itis equally well established that TGFβ remains an inhibitor for ER⁻autonomous cells (Arteaga C L et al. (1988) Cancer Res 48, 3898-3904;Osborne C K et al (1988) Breast Cancer Res Treat 11, 211-219). Drawingfrom the fact that ER⁺ breast cancer cells lack TGFβ receptors (ArteagaC L et al. (1988) Cancer Res 48, 3898-3904; Brattain M G et al. (1996) JMammary Gland Biol Neoplasia 1, 365-372), early onset autonomous breastcancer very likely does not arise from responsive cancer cells, butinstead arises directly from normal cells as outlined in FIG. 123 by theloss of immune surveillance. The term “immune surveillance” means thatcell growth inhibitory immunoglobulins in the general circulation,and/or secreted by or bathing the mucosal/epithelial tissues, arepresent and are in sufficient amounts to deter or prevent cancer cellproliferation. This model has many clinical implications andapplications for diagnosis and genetic screening to identify young womenat greatest risk of developing breast cancer. Early onset markers willbe loss of immune surveillance without obligatory loss of TGFβ effects.The fact that ER⁺ breast cancer cells lack TGFβ receptors while ERbreast cancer cells do have the TGFβ receptor mitigates in favor of thenew bifurcated progression model, in which both ER⁺ and ER⁻ cancersarise directly out of normal breast cells. Because it is statisticallyvery unlikely that an ER⁺ cancer cell, after having lost the TGFβreceptor, would somehow regain that receptor before passing continuingonward to become an ER cancer cell, this non-linear alternative model isreasonable.

Discussion of Example 27

The therapeutic implications of the TGFβ system have been reviewed(Arrick B A (1996) J Mammary Gland Biol Neoplasia 1, 391-397; Reiss Mand Barcellos-Hoff M H (1997) Breast Cancer Res Treat 45, 81-95).However, the model presented in the present Example integrates theinvestigator's discovery of the involvement of the secretory immunesystem with the well known but complex (Koli K M and Arteaga C L (1996)J Mammary Gland Biol Neoplasia 1, 373-380) effects of TGFβ on breastcancer cells. It is expected that a lesion in the genetics or expressionof TGFβ and/or its isoform system of three receptors (Chakravarthy D etal. (1999) Int. J. Cancer 15, 187-194) will have importance inmodulating the estrogen reversible effects of the secretoryimmunoglobulins.

Conversion of normal cells to ER⁺ responsive breast cancers involves theloss of expression of the TGFβreceptor system including one or more ofthe three different forms of the receptor. Changes in these receptors,either individually or in unison are indicated in development of steroidhormone dependent cancers. It is possible that TGFβreceptor II is ofgreatest importance of the three forms (Gobbi H et al. (1999) J NatlCancer Inst 91, 2096-2101). Nonetheless, other studies suggest receptorforms I, II and II as important. As yet, those results have not beenapplied to genetic screening related to ER⁺ breast cancers. According tothe presently proposed model, lesions in the TGFβ system precede lesionsor other types of losses of the receptors for secretory immunoglobulins.The loss of TGFβinhibitory responses may represent the earliest receptorchange identifiable in estrogen responsive breast cancer. The view thatearly onset breast cancer is a failure in immune surveillance and notnecessarily related to TGFβprovides a new focus for genetic screeningand other diagnostic tools.

Prior to the present invention, there has been no report linking theinhibitory effects of TGFβ with the inhibitory effects of the secretoryimmunoglobulins. It has been reported that TGFβ is an immune modulator(Palladino M A et al. (1990) Ann NY Acad Sci 593, 181-187; Letterio J Jand Roberts A B (1998) Annu Rev Immunol 16, 137-161). It is a member ofthe cytokine family, and as such has effects on cells of the immunesystem. It is known that TGFβ has bifunctional effects on mucosal IgAresponses (Chen S-S and Li Q (1990) Cell Immunol 128, 353-361) andinhibits IgG, IgM and IgA production by human lymphocytes (van den WallBake A W et al. (1992) Cell Immunol 144, 417-428). The discovery of thegrowth-regulating role of the immunoglobulins places the complex effectsof TGFβ in a new perspective. Increased TGFβ production can lead tosuppression of the immunoglobulins and therefore positive growth effectson breast cancer cell growth. In the past other investigators have noteda positive effect of TGFβ on breast cancer cell growth under somecircumstances, but had no explanation for this observation (Arteaga C Let al. (1996) Breast Cancer Res Treat 38, 49-56). The results herein nowsuggest a mechanism for TGFβ positive effects on breast cancer cellgrowth. Overproduction of TGFβ is a potential issue that is pertinent tothe growth of estrogen responsive breast cancers.

Example 28 Windows of Breast Susceptibility to Carcinogenesis andMutation and the Levels of Immunoglobulin Inhibitors

In this Example, age-related changes (i.e. a reduction) inimmunoglobulin concentrations in the plasma of rats are correlated withcarcinogenesis of the mammary gland.

“Windows” and Breast Cancer.

Mutations leading to breast cancer may occur early in life, duringpuberty and young adulthood, and control of DNA synthesis by IgA/IgMduring this critical period may attenuate the action of carcinogens andreduce the risk of breast cancer later in life (Marshall E (1993)Science (Wash DC) 259, 618-621). Human female breast cancer incidencerates increase dramatically after age 50 and now approach one in ten byage 75. The existing data suggest that the causal mutations most likelyoccur at earlier ages. In view of the fact that milk/breast secretionsdecrease dramatically after menopause, it remains to be determinedwhether mutations can arise later in life due to the natural age-relatedreduction in the growth inhibitory function of the secretory immunesystem IgA and IgM. An entirely new approach to the prevention of breastcancer is proposed, which includes administering IgA and IgM to youngfemale rats, initially, to diminish the effects of carcinogens byIgA/IgM control of DNA synthesis. These treatments are then followed byoral “immunizations” to increase the natural levels of immunoglobulinsecreting B-cells within the mammary tissue. This new oral immunizationplan is the first attempt to prevent breast cancer by this strategy byenhancing immune surveillance in the individual.

Entry into Phase II—In Vivo Studies with Rats.

The studies described hereinabove were performed in cell culture, andconstitute the Phase I studies. That work employed well-established invitro cell culture models recognized generally to yield physiologicallyrelevant information. Following the in vitro studies, is Phase II, usinganimal models to further define the role of the secretory immune systemin breast cancer etiology and growth in vivo.

Mammary Carcinogenesis Literature Background.

Mammary carcinogenesis in female rodents is most effective during thedevelopmental period that spans early puberty through early youngadulthood (Welsch C W (1985) Cancer Res 45, 341503443; Huggins C et al.(1961) Nature (Lond) 189, 204-207; Janns D H and Hadaway E I (1977) ProcAm Assoc Cancer Res 18, 208; Moon R C (1969) Int J Cancer 4, 312-317;Russo J and Russo I H (1978) J Natl Cancer Inst 61, 1451-1459; Dao T L(1969) Science (Wash DC) 165, 810-811; Meranze D R et al. (1969) Int JCancer 4, 480-486; Haslam S Z (1979) Int J Cancer 23, 374-379; Russo Jet al. (1979) Am J Pathol 96, 721-736; Gullino P M et al. (1975) J NatlCancer Inst 54, 401-414; Grubbs C J et al. (1983) J Natl Cancer Inst 70,209-212). Single challenges with mammary specific carcinogens duringthis time cause tumors in the majority of animals within one year.Similar challenges later during adulthood are far less effective. Theresults of a typical carcinogen experiment with female rats are shown inFIG. 124. Those results show the effects of 3-methylcholanthrene (3MCA)and dimethylbenz[a]anthracene (DMBA). Both carcinogens are commonly usedto induce hormone responsive rat mammary tumors. Carcinogenesis is mosteffective between the ages of 30 days and 100 days, and far lesseffective in rats beyond 150 days. These data support the conclusionthat a “window” exists during which mutations can be induced that leadto breast cancer later in life. There is a strong correlation of this“window” to the timing of “terminal end bud” development in the breasttissue of female rats (Russo I H and Russo J (1978) J Natl Cancer Inst61, 1439-1449). The age relatedness of carcinogenesis in rat mammarygland is paralleled in rat ovary and rat prostate.

There is an expanding body of evidence that indicates that there is a“window” in human females in which the breast is more susceptible tocancer causing changes than at other times in life (Bhatia S et al.(1996) New Eng J Med 334, 745-793; Boice J D and Monson R R (77) New EngJ Med 59, 823-832; McGregor D H et al. (1977) J Natl Cancer Inst 59,799-811; Kaste S C et al. (1998) Cancer 82, 784-792; Boice J D (1996)Med Pediatr Oncol (Supplement 1), 29-34; Cook K L et al. (1990) AJR Am JRoentgenol 155, 39-42; Beaty O III et al. (1995) J Clin Oncol 13,603-609; Shapiro C L and Mauch P M (1992) [Editorial] J Clin Oncol 10,1662-1665). Exposure of 10 to 19 year old human females to ionizingradiation or chemical mutagens (e.g. atomic bomb survivors and patientstreated by chemotherapy and radiation for Hodgkin's disease and othercancers) leads to higher than expected breast cancer rates later inlife. Similar exposures of adult human females were far lessdeleterious. The explanation for these observations is the fact thatmammary gland DNA synthesis increases during puberty and young adulthoodis due to the onset of the differentiation program (Russo J et al.(1982) Breast Cancer Res Treat 2, 5-73) and sex hormone secretion. Asgland terminal end buds develop, they are the sites for mutagenesis(Russo J et al. (1982) Breast Cancer Res Treat 2, 5-73). Clearly, DNAsynthesis is required for carcinogenesis of mammary gland (Welsch C W(1985) Cancer Res 45, 341503443; Gullino P M et al. (1975) J Natl CancerInst 54, 401-414; Grubbs C J et al. (1983) J Natl Cancer Inst 70,209-212; Dao T L (1962) Cancer Res 22, 973-981; Dao T L and Sunderland J(1959) J Natl Cancer Inst 23, 567-581; Dao T L (1981) Banbury Report 8,281-298; Huggins C et al. (1959) J Exptl Med 109, 25-42; Nagasawa H andYanai R (1974) J Natl Cancer Inst 52, 609-610; Sinha D K and Dao T L(1980) J Natl Cancer Inst 64, 519-521; Sinha D K and Pazik J E (1981)Int J Cancer 27, 807-810). It is now proposed that this carcinogenesistiming may be due to changes in the secretory immune system negativeregulation during this critical “window” period.

Correlation of Immunoglobulin Concentrations and Carcinogenesis in RatMammary Gland.

Studies were conducted to demonstrate for the first time that the periodof maximum sensitivity of the mammary gland to carcinogenesis correlateswith times of lowest IgG, IgA and IgM concentrations in the plasma offemale Sprague-Dawley (S-D)-rats. Because all three immunoglobulinclasses are believed to inhibit normal mammary cell replication (TABLE12), an antibody was selected that would identify all three classes ofimmunoglobulins. This choice was rabbit anti-human SHBG, whichrecognizes the three classes of rat Ig that are of interest (FIG. 66).Before initiating these studies, two control studies were done to ensurethat the anti-SHBG obtained from a commercial source (Accurate)effectively recognized all of the growth inhibiting activity in serum.

Immunoprecipitation of the Estrogenic Activity in CDE-Horse Serum andCDE-Rat Serum.

The addition of various dilutions of anti-human SHBG to horse serumeffectively reduced the estrogenic activity of this serum (FIG. 125).The experiments were performed by incubation of the serum with thedesignated dilution of antiserum followed by addition of immobilizedprotein A/G to absorb the rabbit antibody complexes. Each assay startedwith 40% CDE-horse serum. Addition of antibody progressively reduced theestrogenic effect. The results in FIG. 125 show that this was due to aremoval of the inhibitor. A similar analysis was repeated with CDE-ratserum from adult animals >270 days of age. The results are shown in FIG.126. Anti-SHBG effectively neutralized the estrogen reversible inhibitorin serum. Additionally, the studies herein have demonstrated that theactive fraction containing the growth regulating activity binds sexsteroid hormones. To further verify that anti-SHBG was an appropriateantibody, the experiments shown on FIG. 127 were performed. The specificbinding of ³H-DHT to the serum was measured as described (Mickelson K Eand Petra P H (1974) FEBS Lett 44, 34-38), followed by addition ofanti-human SHBG and immunoabsorption with protein A/G. The anti-serumneutralized the labeled steroid hormone binding in both rat and horseserum.

Immunoglobulins in the Serum of Female Rats from Various Age Groups.

FIG. 128 shows that the serum content of the immunoglobulins variedversus age, as determined by Western analysis. FIG. 128 also shows thedensitometry of the Western results with each age group. Initially at 20to 21 days of age (i.e. weaning), the Ig concentrations were at adultlevels. IgG is high immediately after weaning because of gut absorptionand placental transfer from mother's milk. Between days 34 and 60, theconcentrations of total immunoglobulins (i.e. IgG, IgA and IgM) fell by80%. Estrus begins gradually, but is active by day 41 and reaches fulladult expression by 120 days (Döhler K D and Wuttke W (1975)Endocrinology 97, 898-907; Ojeda S R et al. (1976) Endocrinology 98,630-638; Döhler K D and Wuttke W (1974) Endocrinology 94, 1003-1008). At120 days, the immunoglobulin content of the serum was againsubstantially increased. The content was even higher in multiparousretired breeders of >250 days age. Comparison of the results in FIG. 128with those in FIG. 124 indicates that immunoglobulin levels are lowestin rats when carcinogens are most effective. Notably, IgA levels inhuman females are low during childhood and early adolescence, and reachadult concentrations only after 16+ years (Leffell M S et al. (1997)Handbook of Human Immunology, CRC Press, Boca Raton, pp 86-90). Theseobservations suggest that rat and human females have the same “window”with regard to Ig including IgG, IgA and IgM. This set of facts are alsoaddressed in examples that follow.

Discussion of Example 28

This is the first study to correlate changes (i.e. a reduction) inimmunoglobulin concentrations in plasma with carcinogenesis of themammary gland. Continuing Phase II studies will include an animaltesting program to define the specific inhibitory roles of IgG, IgA andIgM in mammary gland growth in vivo.

This study has additional implications. It is well known that mammarygland of multiparous females is resistant to carcinogenesis. In fact,longer-term nursing significantly reduces the risk of breast cancer. Itis also well known that the hormonal environment that accompaniesnursing establishes the secretory immune system in breast. The studiesherein lead to the concept that female hormones or other developmentalchanges increase the content of the secretory system including B cellsin breast tissue. This implies that hormone therapies must be examinedfor effects on the secretory system content of breast. This in turn canbe used to develop new agents and drugs that increase content, and hencereduce the susceptibility of breast to carcinogens or any of many otherpotential mutation causing agents or effects. Further studies aredirected at addressing this issue using carcinogen sensitive adolescentfemale rats, as well as sexually mature females and multiparous females,both of which are more carcinogen resistant than the younger females(Moon R C (1969) Int J Cancer 4, 312-317; Russo J and Russo I H (1978) JNatl Cancer Inst 61, 1451-1459; Dao T L et al. (1960) J Natl Cancer Inst25, 991-1003). The rat mammary tumor model was chosen because of thelarge carcinogenesis data base available (Welsch C W (1985) Cancer Res45, 341503443; Huggins C et al. (1961) Nature (Lond) 189, 204-207; JannsD H and Hadaway E I (1977) Proc Am Assoc Cancer Res 18, 208; Moon R C(1969) Int J Cancer 4, 312-317; Russo J and Russo I H (1978) J NatlCancer Inst 61, 1451-1459; Dao T L (1969) Science (Wash DC) 165,810-811; Meranze D R et al. (1969) Int J Cancer 4, 480-486; Haslam S Z(1979) Int J Cancer 23, 374-379; Russo J et al. (1979) Am J Pathol 96,721-736; Gullino P M et al. (1975) J Natl Cancer Inst 54, 401-414;Grubbs C J et al. (1983) J Natl Cancer Inst 70, 209-212), and theabundance of applicable methodologies. Also, there is convincingevidence that carcinogen induced rat mammary cancers are histologicallysimilar to those of human breast (Russo J and Russo I H (1978) J NatlCancer Inst 61, 1451-1459; Russo J et al. (1982) Breast Cancer Res Treat2, 5-73; Dao T L (1964) Prog Exp Tumor Res 5, 157-216; Russo J et al.(1977) J Natl Cancer Inst 59, 435-445; Murad T and vov Ham E (1972)Cancer Res 32, 1404-1415). Additionally, environmentally relevantcarcinogens (El-Bayoumy K (1992) Chemical Research Toxicology 5,585-590; Wakabayashi K et al (1992) Cancer Res Supplement 52,20922-2098s; El-Bayoumy K et al. (1995) Carcinogenesis 16, 431-434) wereselected for testing the inhibitory roles of IgG, IgA and IgM inattenuating carcinogenic effects. It is noteworthy that lipophilicpolycyclic hydrocarbons such as DMBA and 3MCA and the soluble alkylatingagent NMU effectively transform mammary tissue with single doses (WelschC W (1985) Cancer Res 45, 341503443; Huggins C et al. (1961) Nature(Lond) 189, 204-207; Janns D H and Hadaway E I (1977) Proc Am AssocCancer Res 18, 208; Moon R C (1969) Int J Cancer 4, 312-317; Russo J andRusso I H (1978) J Natl Cancer Inst 61, 1451-1459; Dao T L (1969)Science (Wash DC) 165, 810-811; Meranze D R et al. (1969) Int J Cancer4, 480-486; Haslam S Z (1979) Int J Cancer 23, 374-379; Russo J et al.(1979) Am J Pathol 96, 721-736; Gullino P M et al. (1975) J Natl CancerInst 54, 401-414; Grubbs C J et al. (1983) J Natl Cancer Inst 70,209-212) but are not found in our environment (El-Bayoumy K (1992)Chemical Research Toxicology 5, 585-590; Wakabayashi K et al (1992)Cancer Res Supplement 52, 20922-2098s; El-Bayoumy K et al. (1995)Carcinogenesis 16, 431-434). NMU has been excluded from these studiesbecause it causes specific changes in the ras proto-oncogene (Sukumar Set al. (1983) Nature (Lond) 305, 658-661; Zarbl H et al. (1985) Nature(Lond) 315, 382-385) which are not common in human breast cancers. Ithas been previously suggested that as many as 80 or 90% of human breastcancers are caused by environmental carcinogens (Higginson J (1972) In:Environment and Cancer: 24^(th) Symposium on Fundamental CancerResearch, Williams and Wilkins, Baltimore, pp 69-92; Haenszel W andKurihara M (1968) J Natl Cancer Inst 40, 43-68). To date, however, thisremains to be established.

In this series of studies, DNA synthesis will be monitored in the agegroups spanning 20 days to 270 days. When the period of maximum DNAsynthesis is identified, IgA and IgM compositions will be administered,as injections, to suppress DNA synthesis during this time. After aneffective immunoglobulin dose is found, the appropriate age group willbe treated with IgA/IgM and the effects on carcinogenesis assessedversus control animals. The expected result is that carcinogens will beless effective in those rats receiving DNA synthesis inhibiting doses ofIgA/IgM. In another series of studies, conditions for increasing B-cellpopulations in breast tissue will be determined. To begin, B cellcontent of mammary tissue will be monitored as a function of age. Thiscontrol study will then be correlated with the time period of maximumDNA synthesis. It is expected that the content of B cells will be low inthose age groups showing a maximum DNA synthesis rate. Next, using oralchallenges, it will be determined what is the most effective “immunogen”to induce an increase in B cells in mammary tissue. The end point ofthese studies will be to induce sufficient numbers of B cells to preventthe “window” increase in DNA synthesis. When conditions have beenestablished to prevent this rise, the animal will be treated withcarcinogens and monitored for tumor development and survival. This studyis expected to provide Phase II evidence supporting an oral“immunization” to reduce the effectiveness of carcinogens.

Other ongoing studies will include disruption of the function of thesecretory immune system in adult and multiparous female rats todetermine if they become more sensitive to carcinogens. Virgin femalesof 114 days or older will be studied as will breeders of more than 250days age. These animals will be treated with antibody against thepoly-Ig receptor. The doses of antiserum to disrupt the secretory immunesystem will be established by monitoring IgA/IgM secretion into bile,uterine fluids and breast milk. Also, mammary DNA synthesis will bemonitored. When secretion has been blocked effectively, susceptibilityto carcinogens will be explored. It is expected that the disruption ofthe interaction of IgA/IgM with the poly-Ig receptor will increase DNAsynthesis in the mammary gland and therefore increase susceptibility tocarcinogens. Other ongoing work will determine if mutations leading tobreast cancer occur early in life during puberty and young adulthood andwhether control of DNA synthesis by IgA/IgM during this critical periodwill attenuate the action of carcinogens and reduce the risk of breastcancer later in life.

Example 29 Risk Factors: IgA/IgM Based Test to Detect Lowered Levels ofSteroid Hormone Reversible Cell Growth Inhibitors in Plasma or BodySecretions

IgA/IgM and Cancer Susceptibility.

Toward identifying individuals with high susceptibility to breast canceror prostate cancer, the level of the inhibitory form of IgA (i.e., IgAdimer) will be measured in an individual's plasma, or the secretory IgAand polymeric IgM will be measured in a bodily secretion. Decreases inplasma levels of IgA or decreased secretory capacity into milk orstructural alterations in IgA may confer greater susceptibility tobreast cancer. Levels are expected to be low in susceptible individualsand to fall with increasing age in normal individuals, substantiallymirroring the age distribution pattern associated with breast andprostate cancer incidence. One way to assay for the dimeric/polymericform of IgA is via a conventional antibody binding test using antibodyraised against the D5 domain disulfide regions with IgA attached. Insecretory fluids, direct measure of sIgA can be done along with ameasure of secretory component by radioimmunoassay or other methodsusing enzyme linked immunosorbent assay (ELISA) or biotin-avidintechnology, each of which are well known in the art and have beendescribed in the literature. The levels of IgM can be measured directlyalthough their levels are more subject to wide variations. Also, “J”chain can be measured, but only in samples treated to remove the free(unbound) form known to be in plasma.

Secretory Immune System Status Test.

Another informative test process will be to use rectal or nasal passageantigen challenge and then measure the appearance of the specificantibody against the antigen in plasma and secretory fluids, usingstandard high capacity clinical test methods. This will directly measurethe immune status of the individual. Those with optimum capacity can beseparated from individuals with impaired secretory immune systemfunction. Impaired function of the secretory immune system may indicatesusceptibility to cancer.

Cell Growth Testing for Inhibitors.

In those cases where direct assessment of inhibitor in fluids isrequired, these can also be measured by cell growth assays on reducedmicrowell scale using automated colorimetric assays. The testing iscarried out by first treating a plasma specimen to deplete orsubstantially remove the steroid hormone content without inactivatingor, removing the endogenous poly IgA dimer and poly IgM molecules. Thehormone depleted specimen is then tested for cell growth inhibitoryactivity in the presence of added steroid hormone in an in vitro assayemploying cultured tumor cells incubated in a defined serum-free medium.Procedures for preparing the steroid hormone depleted plasma or serumand for conducting the assay are described in preceding examples and inU.S. patent application Ser. No. 09/852,958 PCT/US2001/15183 entitled“Compositions and Methods for Demonstrating Secretory Immune SystemRegulation of Steroid Hormone Responsive Cancer Cell Growth,” herebyincorporated herein by reference. Application of the XAD-4™ resintreatment is preferred for small samples. These extraction methods arecapable of yielding steroid hormone depleted serum that allowsidentification of 30 to 100-fold estrogen and androgen growth effects(cell number measurement) in culture in 7 to 14 days with human breastand human prostate cancer cells, as well at rat mammary, rat pituitaryand Syrian hamster kidney tumor cells.

Comparison of In Vitro and In Vivo.

The results are compared to similar tests using positive and negativecontrol plasmas or serums, which have defined levels of IgA dimer andpoly IgM. In this way the tumor cell growth inhibitory activity of theindividual's plasma is measured. Because the in vitro assay systememploys a cell line that forms breast or prostate tumors when implantedin vivo, the in vitro assay results are believed to be suggestive of thein vivo condition of the individual.

Discussion of Example 29

Rats and humans process plasma and locally produced IgA verydifferently. This topic is covered in detail (Conley M E and Delacroix DL (1987) Ann Internal Medicine 106, 892-899). In rat, pIgA equilibrateswith locally produced IgA and is therefore a major source of theimmunoglobulin found in secretions. This means the IgA from plasmareadily leaves this compartment to arrive at mucosal surfaces and betransported by the poly-Ig receptor into the lumen of mucosal tissues orinto secretions such as bile. This physiology makes the rat a veryuseful experimental tool to determine some of the cancer related effectsof IgA and IgM. However, caution is necessary when extrapolating ratresults to humans (Conley M E and Delacroix D L (1987) Ann InternalMedicine 106, 892-899). Human plasma IgA (pIgA) does not appear to be asavailable to local tissues for secretion. Indeed, only a small fractionof the secreted IgA in humans comes from plasma IgA. The vast majorityarises locally in mucosal tissues from B cells located there andfunctioning on site. In light of this difference between rat and humanIgA processing, measurement of IgA in the plasma is best approached fromthe IgA deficiency perspective described below. Measurement of thecapacity of the secretory immune system in all subjects by directmeasurement in fluids (e.g. breast fluid, saliva, tears, seminal fluid,bile or vaginal washes) is preferred.

One of the best approaches to measurement of secretory immunoglobulinsin small volumes of body fluids is to challenge with an antigen to whichdifferent low molecular weight haptens are conjugated by standardchemistry now well known and very widely applied. Haptens are conjugatedto common non-antigenic proteins and identified by measuring theappearance of anti-hapten immunoglobulins in the secretory fluids. Bychanging haptens, this test can be administered many times over a periodof years.

Example 30 Risk Factors: IgA Deficiencies and Malignancies

In this Example, measurement of plasma IgA levels are correlated toincreased incidence of mucosal cancers. IgA deficiency is the mostcommon primary immunodeficiency encountered in man (Schaffer F M et al.(1991) 3, 15-44). It is very heterogeneous and is associated withinfections, allergies, autoimmune disorders, gastrointestinal diseaseand genetic disorders. An overview of immunodeficiency-associated cancerhas been presented (Beral V and Newton R (1998) J Natl Cancer InstMonograph 23, 1-6). Breast cancer risk or incidence was not consideredspecifically. Other reports have related this deficiency to abdominalT-cell non-Hodgkin's lymphoma (Ott M M et al. (1998) Am J Surg Pathol22, 500-506; Zenone T et al. (1996) J Intern Med 240, 99-102; FilipovichA H et al. (1994) Immunodeficiency 5, 91-112) and other malignancies(Pongracz K et al. (1994) Orv Hetil 135, 2863-2866). One of the mostsignificant aspects of these reports is the correlation to gastriclymphoma that is currently thought to originate from a bacterial cause.Again, breast cancer and several other mucosal cancers were either notconsidered or were discussed not considered specifically (Butler J E andOskvig R (1974) Nature (Lond) 249, 830-833). Other than the well-knownrelationship between ataxia telangiectasia with its characteristic IgAdeficiency, and breast cancer, there are no other studies of this issueknown to the inventor. This fact also extends to prostate cancers andIgA deficiencies. Measurement of plasma IgA as a measure of propensityto develop breast, prostate and other mucosal cancers is believed to beapplicable for conducting widespread screening programs.

IgM Compensation for IgA Deficiency.

It is of interest to note that IgA deficiency is accompanied by acompensatory increase in IgM (Brandtzaeg P et al. (1968) Science (WashDC) 160, 789-791). Analysis of milk from IgA deficient women indicatessubstantial increases in IgG subclasses and IgM (Hahn-Zoric M et al.(1997) Pediatr Allergy Immunol 8, 127-133; Thom H et al. (1994) AcataPaediatr 83, 687-691). In combined deficiency patients, IgM levels risesufficiently to cause IgM nephropathy (Oymak O (1997) Clin Nephrol 47,202-203). Measurement of plasma IgA, as a tool to determinepredisposition to breast cancer, can be accomplished by standardclinical assays with high specificity antibodies to human IgA, preparedaccording to methods known to those skilled in the art. IgM levels canbe measured similarly.

Example 31 Risk Factors: Autoimmunity Test for Anti-IgA and IgM inPlasma

Methods and immunoglobulin inhibitors described in preceding examplesare useful for conducting studies to identify factors that are capableof neutralizing the IgA/IgM inhibitory effects on cancer cell growth.

General Applicability.

IgA and IgM are estrogen reversible inhibitors of ER⁺ breast cancer cellgrowth in the classical sense of the long sought after chalones. Theyarrest cell growth and are readily reversed within one week in cultureand appear to be mucous epithelial cell specific in function. Theseresults may have implications for epithelial cancers beyond those ofbreast.

Auto-Antibody Properties and Source.

Anti-IgA antibodies purified from normal female plasma will be tested todetermine if they neutralize IgA as a negative growth regulator forbreast cancer cells in serum-free defined culture, employing the cellgrowth assay procedures described hereinabove. These immunoglobulinswill be isolated by standard methods in the literature and their classand subclass determined. They will be fragmented to determine ifactivity resides in the Fab component, as expected in view of theresults described in preceding Examples. Specific blocking monoclonalantibodies will be raised against the active component to permitmeasurement in the serum of females. The purpose of this test is todetermine if an autoimmune mechanism can abrogate the negative IgAgrowth regulation exerted on estrogen responsive breast cancer cells.Such studies will assist in identifying new factors involved in breastcancer etiology. To date, autoimmunity has not been given significantattention with this disease. This study is expected to reopenconsideration of autoimmunity and breast cancer, and a similar approachis applicable to prostate, colon and other mucosal cancers.

Autoimmunity and Cancer.

The concept that autoimmune mechanisms are involved in cancerdevelopment is not new. However, the present findings showing a directcell growth modulating role for the secretory immune system is totallynew. It has been reported that serum from 26 (all) normal volunteers hadanti-IgA antibodies of the IgG and IgM classes (Jackson S et al. (1987)J Immunol 138, 2244-2248). They were purified and were directed againstboth polymeric and monomeric IgA1 and IgA 2 containing the light chains(Fab fragments). Plasma samples will be used to purify similarantibodies, as described above, except in this case, with the goal ofisolation of Fc directed antibodies. The purified antibodies will beidentified by class and fragmented into Fc and Fab portions. Theanti-IgA antibodies will be assessed for their ability to block theaction of IgA as an ER⁺ breast cancer cell growth mediator as described(Sato J D et al. (1987) Methods Enzymol 146, 63-82; Arteaga C L et al.(1988) Mol Endocrinol 2, 1064-1069; Sato J D et al. (1983) Mol Biol Med1:511-529; Gill G et al. (1984) J Biol Chem 259, 7755-7760). Those thatprove effective will be used to raise specific monoclonal antibodies asdescribed (Barret C H (1994) Hybridomas and monoclonal antibodies, In:Antibody Techniques, Malik V S & Lillehoj E P, Eds, Academic Press, SanDiego, pp 71-102). After confirming by double diffusion tests and otheranalyses that the monoclonal antibody recognizes only the appropriateanti-IgA in serum, a RIA will be developed for quantification of serumsamples (Lauritzen E et al. (1994) In: Antibody Techniques, Malik V S &Lillehoj E P, Eds, Academic Press, San Diego, pp 227-258). To establisha control baseline, groups of 100 female serum samples will be obtainedand assays done to establish a basal “normal” range for the blockinganti-IgA antibody. The age and hormonal status of the women donors willbe determined. This will identify a pattern of age differences shouldthey occur. The effects of estrogen containing contraceptives andestrogen replacement therapy will be evaluated. This is especiallyvaluable information because breast cancer occurrence is highly agedependent. Although a naturally occurring antibody has not yet beenidentified that can directly block the growth regulating effect of IgA,its identification will provide a new tool to measure breast cancer riskand risk for other mucosal cancers. This study makes use of several ofthe methods and compositions described hereinabove, includingimmunoglobulin inhibitors compositions, assay methods, defined media andmodel cell lines.

Autoimmune Antibodies.

Alternatively, or additionally, plasma and bodily fluids may bemonitored for autoimmune antibodies that block the inhibitory action ofIgA and IgM. An expected increase in autoimmune antibodies withincreasing age is expected to coincide with increased cancer incidence,or the incidence of cancer may be high in individuals with early onsetdisease.

Example 32 Diagnostic and Prognostic Tools: Estrogen Receptor γ (ERγ)

In this Example, a new estrogen receptor is identified and its role inestrogen responsive cell growth is described. Use of the new ERγ as anadditional or replacement for ERα in gene screening procedures is alsodiscussed.

ERα as the Basis for Most ER Analyses of Breast Cancer Specimens.

In preceding Examples, a new estrogen receptor has been proposed thatregulates estrogen responsive target tumor cell growth. The measurementof this new receptor as a diagnostic and prognostic tool has greatclinical consequences. Currently, throughout the world, the measurementof the known estrogen receptor a (ERα) is accepted as the standard fordetermining whether a breast cancer is estrogen sensitive or estrogeninsensitive (Henderson I C and Patek A J (1998) Breast Cancer Res Treat52, 261-288; Osborne C K (1998) Breast Cancer Res Treat 51, 227-238;Kaufmann M (1996) Recent Results Cancer Res 140, 77-87; Allred D C etal. (1998) Mod Pathol 11, 155-168).

Candidates for the ERγ.

It has been reported that a point mutation in ERα causes it to becomehypersensitive to estrogens (Lemieux P and Fuqua S (1996) J SteroidBiochem Mol Biol 56, 87-91). The point mutation is located in thehormone-binding domain. Growth of the human MCF-7 breast cancer cellstransfected with this point mutation ERα variant is stimulated by 10⁻¹²to 10⁻¹¹M E₂ (Fuqua S A et al. (2000) Cancer Res 59, 5425-5428). Thoseinvestigators proposed that this variant is a point mutation in the ERαthat occurs in premalignant breast tissue lesions. They did not suggestthat it is the growth regulating form of the ER that occurs naturally inall target cells. It should be noted that in the preceding examples,dose-response data have been presented with many cell lines of bothrodent and human origins. In every case, the concentration that causedgrowth was well below the affinity constant of the standard ERα. Thisplainly raises a question about the point mutation variant. It must becommon to every cell type in this disclosure as evidenced by theinformation placed in TABLE 1, TABLE 4 and TABLE 10 and the estrogendose-response data shown in FIG. 3 (MTW9/PL2 cells), FIG. 10 (T47Dcells), FIG. 11 (GH₄C₁ cells), FIG. 12 (H301 cells), FIG. 23B (MCF-7K,T47D and MTW9/PL2 cells), FIG. 92 (MCF-7K cells) and FIG. 100 (T47Dcells). To emphasize again, for this point mutation variant to explainall of the data herein, it must be present in every cell line used inthis disclosure. Furthermore, the investigators identifying the pointmutation variant made, the statement that MCF-7 cells had to betransfected with this variant to become sensitive to one to tenpicomolar concentrations of E₂. The results of the studies herein show,however, that this is simply not the case with MCF-7 cells (FIG. 97) orany of the other cell lines studied. The cells are already sensitive toone picomolar estrogen without any such transfection.

Search for Point Mutation Variant in the Cell Lines Used in thisDisclosure.

PCR will be used to search for the point mutation variant in the celllines listed in TABLE 1. This will provide a definite answer to thequestion of physiological significance. Two outcomes appear mostprobable. First, the point mutation receptor is found in all of the celllines. If so, it will be cloned and transfected into ER⁻ cells todetermine if this reestablishes high sensitivity estrogenresponsiveness, as measured in the cell growth assays described inpreceding Examples. Second, if the point mutation is not found in all ofthe lines, it will indicate that the original authors were correct intheir interpretation that this variant of the ERα receptor ischaracteristic of some premalignant breast cancer lesions and not ofmore general significance. In this case, the above-describeddifferential display methods will be continued to identify the ERγ. Thegeneral domains and functions for each domain of ERα are shown in FIG.129. ERγ is expected to be homologous to ERα but to have changes in thehormone-binding domain and possibly in the transacting function and DNAbinding domains because of activation of growth related genes instead ofthe genes activated by ERα.

Applications of ERγ.

The newly identified ERγ will be used in conjunction with or as areplacement for the current ERα as described above in the variousclinical applications in use today for the diagnosis and prognosticevaluation of breast and other mucosal cancers.

Antagonists of the ERγ.

The action of tamoxifen as an antagonist of the ERγ will find use in theevaluation and treatment of estrogen responsive cancers. Bettertreatment regimes employing tamoxifen can be devised because theclinician can now be better informed about the possible effects of thedrug. Development of more effective or specific antagonists will besought using the recombinant form of the ERγ expressed in estrogeninsensitive cells and in extracts of cells expression the transfectedreceptor.

Example 33 Diagnostic, Prognostic and Treatment Decision Tools: Poly-IgReceptor (or the Poly-Ig Like Receptor)

Definition of the Poly-Ig Receptor.

In this Example, the poly-Ig receptor designation is intended to includethe authentic poly Ig receptor as defined (Kraj{hacek over (c)}i P etal. (1992) Eur J Immunol 22, 2309-2315) or a receptor with very similarproperties as described in this disclosure. The receptor that mediatesthe IgA/IgM cell growth inhibitory effect is likely located onchromosome 1, as described below, although it is to some extent possiblethat it is located on another chromosome but still is a poly-Ig likereceptor with the characteristics outlined in this disclosure.

Diagnostic, Prognostic and Treatment Mode Uses of the Poly-Ig-Receptor.

Breast cancer and other mucosal cancer specimens, including those fromprostate, colon, ovary, uterus, cervix, vagina, kidney and bladder, willbe assessed for the presence of ERα and/or ERγ and for the poly-Igreceptor. The cell surface receptor is preferably located and quantifiedby fluorescence immunohistochemistry after an appropriate fixation(Brandtzaeg P and Rognum T O (1984) Path Res Pract 179, 250-266) or byradioimmunoassay as described for other surface receptors (Tonik S E andSussman H H (1987) Methods Enzymol 147, 253-265). Monoclonal antibodiesagainst the whole poly-Ig receptor, the secretory component or specificdomains can also be used to quantify the receptor (Trowbridge I S et al.(1987) Methods Enzymol 147, 265-279). A variety of new enzyme-linkedimmunosorbant assays (ELISA) are also available and can be applied atvery high sensitivity based on biotin-avidin or chemiluminescencetechnology. The particular method to be applied will be dictated by thetypes of specimens supplied.

Applications of Poly-Ig Receptor Positive Results.

Cancer specimens expressing high levels of poly-Ig receptor and the ERare likely highly differentiated tumors for which there are treatmentoptions. The prognosis for these tumors is thought to be very goodprovided the cancer has not moved to new locations. However, metastasesare a definite negative prognostic indicator. These tumor foci can betreated with a combination of tamoxifen and immunotherapy either asdelivered intravenous immunoglobulins or by a natural boosting mechanismvia “oral immunization” to be discussed below. Long-term exposure toboth tamoxifen and IgA/IgM is a new non-toxic approach to treatingdisseminated cancer. Currently, disseminated breast (and other) cancersare treated by chemotherapy or possibly with radiation.

Applications of Poly-Ig Receptor Negative Results.

Cancers not expressing the poly-Ig receptor must still be assessed forER and the progesterone receptor. If ER positive, an appropriatetreatment option is tamoxifen or adjuvant chemotherapy. Even thoughtamoxifen has activity mimicking the immune system inhibitors, it alsohas activity against the ER (which accounts for its classification as a“mixed” antiestrogen). Immunotherapy will not be effective with thesetumors. Cancers that are both poly-Ig receptor negative and ER negativeare expected to have poor prognosis. The best treatment optionscurrently are limited to chemotherapy or in some cases therapy withmonoclonal anti-HER/NEU. This latter treatment has proven to be oflimited application.

Clinical Studies of Secretory Component (Poly-Ig Receptor) Expression inColon and Breast Cancer.

Others have conducted a study of the protein and mRNA expression of thepoly-Ig receptor has been done with a sample of human colon cancers(Kraj{hacek over (c)}i P et al. (1996) Br J Cancer 73, 1503-1510). Inthat study, expression of secretory component was found in 33 colorectaladenomas (31 patients) and in 19 colorectal carcinomas from 19 patients.Although the study provides evidence that colon adenomas (i.e. apredisposition to colon cancer) and confirmed cancers express poly-Igreceptor, the investigators did not attempt to translate theobservations further to than to propose a role in “cellular dysplasia”.

Likewise, the levels of secretory component were measured in breasttumors from 95 patients with primary or metastatic disease (Stern J E etal. (1985) Cancer Immunol Immunother 19, 226-230). The authors of thatstudy proposed that low levels of secretory component were found inmetastatic lesions and that this “could indicate a potential forsecretory component/poly-Ig receptor involvement in immune regulation oftumor growth”. However, neither the identification of growth effectsrelated to the immunoglobulins IgA/IgM nor the identification of a roleof the poly-Ig receptor directly was investigated. That study was alsoincomplete in that there was no attempt made to determine the estrogenreceptor status of the primary or metastatic disease. Therefore, therewas no correlation to growth state based on the most accepted criterionof steroid hormone receptor status. This line of study appears to havestopped with 1985 observation. The present series of studies hasdirectly addressed the problem, however, by demonstrating growthregulation by the secretory immune system using several different ER⁺cancers. These results change the context of the diagnostic analysis ofsecretory component or poly-Ig receptor.

Example 34 Diagnostic Tools: Monoclonal Antibodies to the Poly-IgReceptor and Breast Cancer Imaging

A two-fold approach to breast cancer imaging has been devised thatincludes immunoglobulin directed and poly-Ig receptor directed methods.

Current Imaging Methods.

Today, X-ray mammography remains the most important method for breastcancer screening (Sabel M and Aichinger H (1966) Physics in Medicine andBiology 41, 315-368). Since the 1980s, ultrasound scanning has evolvedas an indispensable adjunct to X-ray mammography. Other procedures suchas Doppler sonography, diaphanography, contrast enhanced MRI, CT and DSAessentially depend upon the enhanced vascularity of the tumor comparedto the surrounding normal tissue. In addition to those methods, computerassistance is used for signal processing which aids diagnosis by textureanalysis and pattern recognition. Along with those methods, scintigraphybased on receptors located in the breast tumors has become a newnon-invasive modality (Valkema R et al. (1996) J Cancer Res Clin Oncol122, 513-532). The presently disclosed method depends upon expression ofa specific receptor, the poly-Ig receptor, in the breast tumor.Monoclonal antibodies to the poly-Ig receptor and/or IgA or IgM (wholemolecule or fragments) will be used to image breast tumors at an earlystage of development.

Poly-Ig Receptor Directed Methods.

Breast and prostate cancer cells bind polymeric IgA and IgM, but incontrast to normal cells, the cancer cells no longer transport theimmuno globulins because of disruption of tissue architecture and lossof baso-lateral orientation required for secretion of theimmunoglobulins. As a consequence, the immunoglobulins accumulate in thecells and are partially degraded with time. When the immunoglobulins areradio labeled or contrast labeled, the markers accumulate in the cancercells compared to the amounts in the surrounding normal cells. Thecancer cells are expected to image at very early stages due to theaccumulation of the tracer or contrast agent. Most breast and prostatecancers begin at the 1 to 2 mm tumor size, so imaging with theIgA/IgM/poly-Ig receptor should be very sensitive. Consequently, thesemethods constitute a significant improvement over existing imagingsystems. Many of the limitations inherent in each imaging methodoutlined above will be present even with the use of IgA, IgM or poly-Igreceptor technology. Nonetheless, the knowledge base available forimaging supports the use of labeled IgA/IgM/poly-Ig receptor as animprovement because the target will be very early stage tumors readilyrecognized by this, technology. Monoclonal antibodies will be prepared,and radio labeled or contrast labeled IgA/IgM and receptor will beprepared, using suitable conventional methods and techniques that arewell known to those of skill in the art.

Example 35 Diagnostic, Prognostic and Treatment Decision Tools: Fc-LikeReceptor for IgG1/IgG2

In this Example, the term “Fc-like receptor” is intended to mean amember of the Fc-superfamily of immunoglobulin-like receptors, possiblywith an inhibitor ITIM motif, as described above.

Diagnostic, Prognostic and Treatment Mode Uses of the Fc-Like Receptor.

Of the mucosal cancers examined, evidence is presented herein forinhibitory effects of IgG on only breast and prostate cells. It islikely that IgG1 and IgG2 will have effects on other early mucosalcancers. Breast cancer and prostate cancer specimens will be assessedfor the presence of ERα and/or ERγ and for the Fc-like receptor. Thecell surface receptor is preferably located and quantified byfluorescence immunohistochemistry after an appropriate fixation(Brandtzaeg P and Rognum T O (1984) Path Res Pract 179, 250-266) or byradioimmunoassay as described for other surface receptors (Tonik S E andSussman H H (1987) Methods Enzymol 147, 253-265). Monoclonal antibodiesagainst the whole receptor or specific domains can also be used toquantify the receptor (Trowbridge I S et al. (1987) Methods Enzymol 147,265-279). A variety of new enzyme-linked immunosorbant assays (ELISA)are also available and can be applied at very high sensitivity based onbiotin-avidin or chemiluminescence technology. The method to be appliedwill be dictated by the types of specimens supplied.

Applications of Fc-Like Receptor Positive Results.

Cancer specimens expressing high levels of Fe-like receptor and the ERare likely differentiated tumors. The prognosis for these tumors isexpected to be very good. These tumors can be treated with a tamoxifenand immunotherapy delivered as either intravenous immunoglobulins or bya natural boosting mechanism via “oral immunization,” which is discussedin an Example that follows. Long-term exposure to both tamoxifen andIgG1κ is a new non-toxic approached to treating these cancers, asindicated by results of studies described in Examples 20 and 24,employing an in vitro model assay system that is useful as an aid forpredicting in vivo effects of a given stimulus, such as a chemical ofinterest.

Applications of Fc-Like Receptor Negative Results.

Cancers not expressing the Fc-like receptor will also be assessed for ERand the progesterone receptor. If ER positive, the preferred treatmentoptions are, for example, tamoxifen or adjuvant chemotherapy. Eventhough tamoxifen has activity mimicking the immune system inhibitors, itstill has activity against the ER (which accounts for its classificationas a “mixed” antiestrogen). Immunotherapy is not expected to beeffective with these tumors. Cancers that are both Fc-like receptornegative and ER negative are expected to have poor prognosis. Thisdiagnostic test should indicate selection of a very aggressivechemotherapy or other program.

Example 36 Diagnostic, Prognostic and Treatment Decision Tools: TGFβReceptors

In this Example, use of TGFβ in detecting early onset breast cancer andfor assessing the status of a tumor is described.

TGFβReceptors.

The TGFβ receptors to be monitored will be isoforms Type I, Type II andType III also designated RI, RII, and RIII as described (Gobbi H et al.(1999) J Natl Cancer Inst 91, 2096-2101; Chakravarthy D et al. (1999)Int J Cancer 15, 187-194). Although breast cancers express all threeforms of TGFβ receptors, only one of these (i.e. TGFβ RIII) has beenlocalized to a “hot spot” for breast cancer on the short arm ofchromosome 1 (i.e. 1p33-p32). Prior art studies of TGFβ expression inbreast cancer specimens have problems based on the fact that it is notclear which cell types in the tissue in fact have the receptors. Becauseclinical specimens are mixtures of cells, methods should be consideredthat establish that the target epithelial cells are either receptorpositive or negative. Immunohistochemistry of fixed tissue is thepreferred method to examine this issue. Appropriate methods have beendescribed (Gobbi H et al. (1999) J Natl Cancer Inst 91, 2096-2101).Based on that study (Gobbi H et al. (1999) J Natl Cancer Inst 91,2096-2101), the Type II receptor is most associated with breastepithelial hyperplastic lesions that increase the risk of laterdevelopment of invasive breast carcinoma. In tumor systems, Type IIreceptor is positively associated with TGFβ responsiveness (i.e. growthinhibition). As the matter stands however, the 3p22 loci for Type IITGFβ receptor (Mathew S et al. (1994) Genomics 20, 114-115) has not yetbeen mapped as a “hot spot” for breast cancer.

Diagnosis of Early Onset ER⁻ Breast Cancer.

Early onset breast cancers can be classified by measurement of their ERcontent, the content of TGFβ receptors (particularly Types II and III)and the poly-Ig receptor/Fc-like receptor. Together, these assessmentsare expected to act as aids to define the cancer type for therapydecisions. While these cancers are expected to be TGFβ receptorpositive, therapy with this 25 kDa inhibitor alone has not beeneffective in the past. These tumors may require aggressive treatmentwith available tools such as standard chemotherapy or high-dosechemotherapy coupled with bone marrow transplant.

Diagnosis of Early Onset ER⁺ Breast Cancer.

However, the methods outlined above can also be used to aid in theclassification of the approximately 30% of the early onset tumors thatare ER⁺. These tumors are expected to be TGFβ receptor negative.Screening for poly-Ig receptors/Fc-like receptors plus the ERα or ERγwill indicate the use of the combined tamoxifen (and/or newer SERMs) andimmune therapy described above. Advantages of this modality are the lackof severe side effects, as well as preservation of fertility, which isoften a major consideration.

TGFβReceptors and ER⁺ Cancers.

Although this discussion has been focused on breast cancer, the samescreening methods are expected to be applicable to a number of other ER⁺types of cancers. As shown in FIG. 26, all of the ER⁺ cell lines testedappeared to be unaffected by TGFβ although data presented throughoutthis disclosure shows these same lines are IgA/IgM inhibited. As canbest be appreciated by referring to the cancer progression model of FIG.123, the combination of positive results with the ER (ERα or ERγ) andpoly-Ig receptor (or Fc-like receptor), along with negative results forTGFβ receptor(s) is a defining pattern for the early breast cancers thatwill be immune system treatable.

Example 37 Ataxia Telangiectasia as an Example of a Human GeneticDisorder with High Rates of Breast Cancer Coupled with an IgA Deficiency

In this Example, analogies are drawn between the characteristic IgAdeficiency in the genetic disorder ataxia telangiectasia (A-T) and therole of IgA in inhibiting steroid hormone responsive cancer growth inmucosal tissues. Homozygotes have high rates of breast cancer (Olsen J Het al. (2001) J Natl Cancer Inst 93, 121-127; Swift M (2001) J NatlCancer Inst 93, 84-85), even in males. Even heterozygotes have highbreast cancer rates (Janin N et al. (1999) Br J Cancer 80, 1042-1045;Inskip H M et al. (1999) Br J Cancer 79, 1304-1307; Lavin M (1998) BrMed J 317, 486-487; Athma P et al. (1996) Cancer Genet Cytogenet 92,130-134; Chen J. et al. (1998) Cancer Res 58, 1376-1379). The mutatedgene is thought to code a product similar to the PI-3 kinase (Savitsky Ket al. (1995) Science (Wash DC) 268, 1749-1753). However, 75% of A-Tindividuals have IgA absent or deficient. Studies have shown that theA-T lesion is not found in breast cancers (FitzGerald M J et al. (1997)Nature Genet. 15, 307-310; Bebb D G et al. (1999) Br J Cancer 80,1979-1981; Vorechovsky I et al. (1996) Cancer Res 56, 2726-2732). Thishas perplexed researchers and suggests that the high risk of breastcancer in A-T individuals may be due to factors other than the reportedgenetic lesion. Secretion of immunoglobulins by mucosal cells iscertainly impaired (Bordigoni P et al. (1982) Lancet 2(8293), 293-297;Boder E (1975) Birth Defects Orig Artic Ser 11, 255-270). Very early on,clinicians noted frequent mucosal infections in A-T individuals.

Based on the results of the studies herein, which establish the role ofIgA in mucosal/breast cell growth, it seems reasonable to suggest thatthe IgA deficiency in A-T has a direct effect on malignancy developmentin mucosal tissues, particularly breast. It is noteworthy that A-T hasbeen discussed often among breast cancer researchers as a model for theetiology of this disease, and was addressed in an editorial (Swift M(2001) J Natl Cancer Inst 93, 84-85). Tests assessing the level andactivity of IgA in an individual, according to an above-described cellgrowth assay method, can be useful for correlating to the presence ordevelopment of malignancy.

Example 38 Diagnostic and Predictive: Poly-Ig Receptor, the Fc-LikeReceptor And TGFβ Receptors Based Genetic Screening for Breast, Prostateand Other Mucosal Cancer Susceptibility

The mediating receptors for IgA/IgM and IgG1 inhibition, identified asdescribed in foregoing Examples, and the TGFβ receptor, will be usefulfor screening individuals for susceptibility to cancer, and for genetherapy applications to restore immune regulation in autonomous tumors.

Background Genetic Properties of the Poly-Ig Receptor.

The complete genomic and cDNA sequences of the poly-Ig receptor havebeen determined (Kraj{hacek over (c)}i P et al. (1991) Hum Genet. 87,642-648; Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22,2309-2315). Poly-Ig receptor gene has been localized to chromosome 1 at1q31-q42 locus Kraj{hacek over (c)}i P et al. (1991) Hum Genet. 87,642-648; Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22,2309-2315; Kraj{hacek over (c)}i P et al. (1995) Adv ExpMed Biol 371A,617-623). The long arm of chromosome 1 had initially been described asthe location of the most frequent ctyogenetic abnormalities found inhuman breast carcinoma (Bieche I et al. (1995), Clin Cancer Res 1,123-127). More recently this conclusion was modified state that distalalterations of the short arm of chromosome 1 are the most frequentcytogenetic abnormalities in human breast carcinoma (Bieche I et al.(1999) Genes Chromosomes Cancer 24, 255-263). The gene encoding thepoly-Ig receptor is linked to D1S58 on the long arm of chromosome 1(Kraj{hacek over (c)}i P et al. (1992) Hum Genet. 90, 215-219). Thislocus (i.e. D1S58) is a known site for “allelic imbalances” in aremarkable 75% of all breast cancers (Loupart M-L et al. (1995) GenesChromosomes Cancer 12, 16-23). Allelic imbalances include “Allelic Loss,Allelic Gain, and Imbalances”. Loss of herterozygosity (LOH) isconsistently high along the length of the long arm of chromosome 1 atD1S58 (i.e. 46%) in breast cancers (Loupart M-L et al. (1995) Genes,Chromosomes & Cancer 12, 16-23). LOH is strongly associated withdevelopment of cancer. The observations in this disclosure now bringmeaning to this published observation. The report describing changes inD1S58 did not specific what gene or type of gene or function might beimpaired by damage to this locus (Loupart M-L et al. (1995) Genes,Chromosomes & Cancer 12, 16-23). The Inventor's results indicate this“hot spot” is either the authentic poly-Ig receptor acting in its newcapacity as a growth regulator, or a very closely related receptor withsimilar molecular weight, ligand binding and immunological properties.However, it must be recognized that the functional form of the growthregulatory receptor may arise from alternate splicing of the poly-Igreceptor gene. Alternate splicing of the poly-Ig receptor gene is knownin rabbit (Deitcher D L and Mostov K E (1986) Mol Cell Biol 6,2712-2715; Frutiger S (1987) J Biol Chem 262, 1712-1715) and bovinetissue (Kulseth M A et al. (1995) DNA Cell Biol 14, 251-256). It has yetto be proven (or disproved) in humans. Certainly this possibility isstill open with hormone responsive cancer cells. Alternately the1q3′-q41 region of chromosome 1 contains several other genes ofimmunological interest (Kraj{hacek over (c)}i P et al. (1991) Hum Genet.87, 642-648; Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22,2309-2315; Bruns G A P and Sherman S L (1989) Cytogenet Cell Genet. 51,67-77). As shown in FIG. 130, the locus of the poly-Ig receptor (PIGR)is distant from the major other loci for breast cancer locatedconchromosome 1. The Entre Genome NCBI Search listed 31 “hot spots” formutations occurring in breast cancer specimens. None of these genes wererelated to the poly-Ig receptor. An expanded diagram of chromosome 1 isshown in FIG. 131. It further emphasizes the fact that the locus of thepoly-Ig receptor will represent a new discovery as a breast cancer gene.There can be little doubt that the discovery herein of immune negativeregulation of growth mediated by the poly-Ig receptor, or one veryrelated, is an advance. It was arrived at not by the genetic approachdescribed above which screens genes without regard for function, butinstead by a functional approach based on the biochemical, endocrine andcell biology studies described above.

Identification of the Poly-Ig Receptor by cDNA Sequencing.

Molecular cloning and cDNA sequencing of the IgA/IgM inhibitionmediating receptor has been generally described in a preceding Example.Preferred ways of carrying out those procedures for identifying thepoly-Ig receptor are provided next. The complete cDNA sequence of thepoly-Ig receptor will be established by PCR cloning or cDNA cloning withantibody screening as described in TABLE 13. This will be done with ER⁺T47D human breast cancer cell lines and the LNCaP prostate cancer cellline. These lines were chosen because they express either the authenticpoly-Ig receptor or one very similar as determined by antibody blockingactivity (FIGS. 114 and 115). Also, by Western analysis the LNCaPcellsexpress an anti-secretory cross-reacting band of the same molecularweight as authentic poly-Ig receptor from HT-29 cells (FIG. 116). Thissame technology will be used to obtain the Fc-like receptor from thesame two human cancer cell lines because they were shown to beresponsive to IgG1/IgG2 (FIGS. 120, 121, and 122). Two strategies appearuseful for this procedure and summarized in (TABLE 13). The selection ofthe appropriate strategy depends upon the results of the studiesoutlined above. For example, if a poly-Ig like or an Fc-like receptor issought, there is sufficient sequence data available to apply PCRcloning. If an entirely new receptor is expected from the receptorbiochemistry studies, cDNA cloning will be required with antibodyscreening. PCR cloning will be done according to published detailedprocedures (Current Protocols in Molecular Biology, Volume 3, (2000)Sections 15.6 & 15.7, cDNA Amplification Using One Sided (Anchored) PCRand Molecular Cloning of PCR Products). The cDNA cloning will be donewith the Lambda TriplEx® Phagemid which gives a three fold greaterchance of finding positive plaques with antibody. The complete manualfor cloning and use of this vector has been obtained by Internet fromClonTech, January 1996 CLONTECHNIQUES.

TABLE 13 Molecular Cloning Strategies for the Poly-Ig Receptor and theFc-like Receptor STRATEGY 2: cDNA CLONING/ STRATEGY 1: PCR CLONINGANTIBODY SCREENING 1. Prepare poly (A)⁺ RNA 1. Identify inhibitoryreceptor blocking antibody 2. Use oligo (dT) primer and RT to 2. Preparepoly (A)⁺ RNA make cDNA 3. Use cDNA with specific primer 3. Use oligo(dT) primer and RT plus oligo (dT) primer to amplify to make cDNA withTaq DNA Polymerase 4. Amplify again with cDNA and 4. Methylate, makedouble internal primers plus oligo (dT) with stranded DNA Taq DNAPolymerase 5. Check size by agarose gels 5. Select Vector (TriplEx in akit) (single band) 6. Clone into AT vector for DNA 6. Ligate DNA intovector sequencing 7. Primers selected with on-line 7. Introduce vectorinto E. coli computer assistance 8. Screen with blocking antibody 9.Amplify clones for DNA sequencing 10. Because the 5′-end may be missing,use Rapid Amplification of cDNA ends (5′-RACE) kit (Ambion) to get afull length clone.

Receptor Identification and Chromosome Localization.

The molecular cloning of both receptors will provide structuralidentification and determine if the poly-Ig receptor is the authenticform previously associated with only transcytosis, or whether it is analtered form. The sequencing results are expected to resolve thealternate splicing issue discussed above. If the sequence resultsindicate a new receptor, chromosomal localization will be done todetermine if it is within the D1S58 linked locus on chromosome 1 orpossibly on another chromosome. If it is located on another chromosome,this will be solid evidence of a new Ig superfamily receptor gene thatnegatively regulates growth. The same discussion applies to the Fc-likereceptor. It is expected that the Fc-like receptor will be a new genebecause of the data showing localization of the other known forms toleukocyte series cells (TABLE 11). Additionally, the amino acidsequences deduced will be used to match to known ITIMs to determinewhether the inhibition regulating receptors are members of this newclass of inhibitory receptors, as discussed above.

Transfection Studies to Regain Immune Regulation and Steroid HormoneResponsiveness.

One ER⁻ cell line will be selected for transfection based on Westernanalysis demonstrating a lack of receptor expression. Also, the DU145cells and ALVA-41 human prostatic carcinoma cells will be used. Thesecell lines are AR⁺ but are not inhibited by immunoglobulins (FIG. 117).Transfection of these cells is expected to restore IgA/IgM inhibitionand possibly permit demonstration of androgen reversibility. If this isidentified, it is very strong evidence for the positive/negative modelproposed herein as the control mechanism for steroid hormone sensitivecells. For the transfection studies, receptor cDNA will be subclonedinto a mammalian cell expression vector. A vector with a CMV promoterwill be used because of its wide range of tissue expression and highlevels of product. This will include a six amino acid sequence of c-myconcogene to detect transformants. This tag will allow the laboratory todistinguish between low levels of endogenous expression and expressiondue to transformation. The transfections will be done with cationicdetergents. This protocol will use the Green Flourescent Protein (GFP)reporter (CMV promoter) which can be visualized directly withoutfixation or staining. Transient expression of the receptor will bemonitored for 80 hours by c-myc immunodetection. To measure the growthinhibitory effects of the IgA or IgM during this time, tritium labeledthymidine incorporation into DNA will be measured. For longer-termstudies, stability-transformed cells will be selected using theantibiotic neomycin and G418. Stable transfectants will be monitored forreceptor expression as described above. If stable transfectants regainimmune control, this will be reasonable support for the conclusion thatan effective receptor has been identified. This is an importantprecursor study for the use of the receptors in gene therapy of breastand prostate cancers a well as other mucosal cancers.

Site Directed Mutagenesis to Identify Critical Domains.

Transfection with the tissue culture models above will be used toidentify and/or confirm domains in which mutations cause loss of thereceptor function. This is an important control because all genes havevariations that may or may not be critical. This has certainly been trueof BRCA1 (Iau P T et al. (2001) Eur J Cancer 37, 300-321). Standard sitedirected mutagenesis methods are planned to alter specific amino acidsor parts or all of selected domains. These cell culture studies will bematched to the sequences being derived from non-disease females todefine natural variations that have no effect versus changes that aresignificant. In the case of BRCA1, the presence of a specific mutationin families with breast/ovarian cancer was used as an importantindication of changes that were significant (Iau P T et al. (2001) Eur JCancer 37, 300-321).

Predictive Genetic Analysis: Germ Line Mutations.

Women with family histories of breast cancer especially in first-degreerelatives are candidates for genetic analysis of the poly-Ig receptorand/or the Fc-like receptor. These analyses will rely on the knowledgeof the important domain or other mutations that have been defined bymonitoring women with breast cancer versus those without disease as wellas information gained above by site directed mutagenesis. Theavailability of a direct biological assay of receptor function versusmutation position and/or type is a distinct advantage over the situationwith BRCA1 and BRCA2 (Iau P T et al. (2001) Eur J Cancer 37, 300-321).The methodology is well described (Malkin D et al. (1990) Science (WashDC) 250, 1233-1238). Skin bioposy fibroblasts or blood leukocytes areextracted to obtain DNA. Using PCR, selected exons will be amplified andDNA sequenced. Multiple primers can be used to cover the whole receptor,especially if it is similar to the eleven-exon structure of the poly-Igreceptor. Generally, Fc-like receptors are >70 kDa, indicating evenfewer exons. Both DNA strands will be amplified. As technology develops,the traditional slab-gel electrophoresis analysis will preferably bereplaced with high throughput mutation screening using automatedcapillary electrophoresis (Larsen L A et al (2000) Comb Chem HighThroughput Screen. 3, 393-409). This will facilitate commercialscreening of large numbers of DNA samples. A significant mutation in oneallele is a potential predisposing factor based on the need for only oneadditional “hit” to have a loss of a critical receptor. These samechanges may be applicable to prostate, colon and other mucosal cancers.

Predictive Genetic Analysis: Other Allelic Imbalances.

There are a variety of other potential genetic changes that maypredispose women to breast cancer. Changes that are especially relevantto this disclosure include loss of heterozygosity (LOH), concomitantgain and loss of alleles (GAL) and simple gain of alleles (GCN) (LoupartM-L et al. (1995) Genes Chromosomes Cancer 12, 16-23). The effect ofeach of these is to increase genetic instability and contribute tochanges that affect the expression of the gene product. These will befurther addressed below in Examples related to tumor diagnostics. Thesesame changes may be applicable to prostate, colon and other mucosalcancers.

Predictive Genetic Analysis: Expression Genetics in Cancer.

One of the most interesting facts of cancer is that relatively few havebeen directly related to mutated genes in humans (Sager R (1997) ProcNatl Acad Sci USA 94, 952-955). What is far more common is thatexpression of genes is changed. The definitions of the different typesof changes are “Class I genes” that are mutated or deleted at the DNAlevel, and “Class II genes” that are not altered at the DNA level butare changed in expression level. In this disclosure, both types ofchanges are included for the poly-Ig receptor (or poly-Ig-like receptor)and the Fc-like receptor. The information gained from characterizingthese changes will be used to improve the diagnosis, prognosis,treatment or prevention of mucosal cancers.

TGFβ Receptors and Genetic Analysis.

The protocols just described above for application to the poly-Igreceptor and the Fc-like receptor are also applicable to, and are herebyextended to include, the TGFβ Type I, Type II and Type III receptorswith breast and prostate cancer, preferably. It can readily appreciatedthat similar analyses can be applied to other mucosal cancers as theyare proven to be regulated by IgA/IgM. The genetic analysis of Class Iand Class II changes in TGFβ receptors will preferably be done incombination with evaluations of the status of ERα and/or ERγ and theimmunoglobulin receptors, as an aid in selection of the most appropriatetherapy for a particular patient.

Primary Tumor Analysis.

Primary tumors will be screened for allelic imbalances as described(Loupart M-L et al. (1995) Genes Chromosomes Cancer 12, 16-23). Based onthe known allelic imbalances associated with breast cancer and locusD1S58, these will be preferred analyses. Other analyses such aschromosomal loss and chromosomal rearrangements are recognized asimportant aspects of cancer development and progression (Lengauer C etal. (1997) Nature (Lond) 386, 623-627) and will be included as receptoridentification loci are defined.

Molecular Assessment of Cancer.

There are several major advances in cancer genetics arising from thepresent invention that promise a new clinical future for cancerdiagnosis, genetic screening, prevention and therapy. These include: (1)A detailed definition of the genetic (DNA) changes and altered geneexpression will become available for mucosal cancers and will includethe new receptors disclosed herein. (2) Obtaining the genetic profile ofa single patient's primary tumor will become a routine matter and permitfar better design of treatment for mucosal cancers. (3) Large scalepopulation based screening will become a reality with samples obtainedby non-invasive procedures or from easily assessable body fluids such assaliva, sputum, urine and mucosal washings. Representative applicationsof these concepts and approaches are described herein. (4) A molecularanalysis of surgical margins and lymph nodes and metastases will becomeroutine, particularly for mucosal cancers, as evidenced herein. (5) Theinformation provided in the present disclosure, and the tools andmethods developed and described herein will be of especial value whenapplied to the preinvasive and preneoplastic states of mucosal cancersbefore they become symptomatic.

Example 39 Breast Cancer Prevention with Applications to Prostate Cancerand Other Mucosal Cancers

Oral immunization strategies have been devised to reduce the risk ofand/or prevent breast cancer and cancers of other mucosal tissues.

World-Wide Breast Cancer Death Rate by Country.

When expressed by death rate per 100,000 population, it is clear thatthe ranking (1 highest and 44 lowest) is highest in industrial/developedcountries of North America and Northern. Europe (TABLE 14). Large Asianpopulations are at the bottom of the ranking. The conventional wisdom isthat the populations of high-ranking areas are exposed to moreenvironmental carcinogens and mutagens, and also have the highestdietary caloric and fat intake. This has led to the general acceptanceof the idea that diet and environment cause breast cancer.

TABLE 14 World-Wide Death Rates for Breast Cancer Deaths per 100,000COUNTRY/RANK Denmark/1 Ireland/2 Netherlands/3 Israel/4 United Kingdom/5Hungary/6 New Zealand/7 Germany/8 Trinidad & Tobago/9 Canada/10Solvenia/11 Czech Republic/12 Austria/13 United States/14 Australia/15France/16 Norway/17 Lithuania/18 Estronia/19 Croatia/20 Republic ofMoldova/21 Portugal/22 Spain/23 Latvia/24 Finland/25 Sweden/26 Greece/27Russian Federation/28 Poland/29 Macedonia/30 Bulgaria/31 Romania/32Cuba/33 Kazakhstan/34 Chile/35 Venezuela/36 Kyrgyzstan/37Turkmenistan/38 Mexico/39 Columbia/40 Mauritius/41 Azerbaijan/42Japan/43 China/44 Slovakia - no data

Comparisons of the World-Wide Death Rates for Colon/Rectal, Breast andProstate Cancer.

Of the major mucosal cancers, colon/rectal, breast and prostate are themost common and have high mortality in many countries. FIG. 132 shows alisting from the World Health Organization (1999) of the deaths per100,000 in 45 countries. Although the correlations are not ideal, thegeneral conclusion is that several of the high ranked countries haveabove average rates of all three types of cancer. These countries againtend to be the industrialize/developed with the dietary andenvironmental problems associated with higher standards of living. Thesestatistics show that mucosal cancer is a common problem in more affluentcountries and that prevention is a major problem that has significancein broad areas of the world.

Plasma Immunoglobulins and Age in Humans.

It is well recognized that during the first few months of life, theimmune system of the infant has not yet developed. The immunoglobulinsin the child's blood are from the mother and are predominantly IgGsubclasses IgG1 and IgG2 (FIG. 133). As shown in FIG. 133, IgM is thenext Ig to increase as early as in the first year. This rise is requiredfor the development of the full immune response. Notably, IgA is muchslower to reach adult levels and only achieves this after age 10+. Thelate appearance of plasma IgA is paralleled in some of the mucosaltissues. Reproductive system mucosal immunity of males and females ishormone dependent and does not develop until the onset of puberty, andthen only reaches adult levels well after this time. This indicates thatduring the period of development of the breast adolescent females, thesecretory immune system is just developing. This is the “window” ofopportunity for mutation described above. If this window were reduced,or its open period decreased, a significant reduction in breast cancerrisk could be expected.

Prevention of Breast and Prostate and Other Mucosal Cancers by “OralImmunization”.

Development of a broadly applicable immunization approach to preventmucosal cancers is urgent. Today, there is no such immunization method.In the present Example, the observations and data presented aboveestablishing the inhibitory effects of the secretory immune system areextended to the development of an oral immunization method based oninduction of increased immunoglobulins in mucosal tissues. This increaseis expected to slow DNA synthesis and thereby reduce the effect ofmutagens during the adolescent female “window”. Furthermore, there isanother “window” caused by menopause. At this time, the secretory immunesystem of breast decreases. This reduces available inhibitors. Existingpreneoplastic cells are no longer under sufficient negative control. Itis proposed that this natural process is a major contributor to thesharp rise in breast cancer incidence after menopause.

Stimulation of the Body's Natural Immune System to Close “Windows”Periods of Mutagen Susceptibility—Dual Benefits.

Breast cancer will be used as a model of mucosal tissues, employing anew approach to preventing or reducing the risk ofbreast/prostate/mucosal cancer by stimulating the body's natural mucosalimmune defense system, preferably via oral immunogens, to prevent earlymutations that ultimately lead to cancer later in life. Evidencepresented herein shows that longer-term exposure of ER⁺ breast cancercells to IgA or IgM will result in cell death within a few weeks inculture. Even given that this process will take longer in vivo, use oforal immunization throughout adult life promises benefits. Byapproaching oral immunization from this perspective, it becomes bothprevention and therapy.

Gastrointestinal Immune System.

It is now proposed that “oral immunization” can be administered to menand women of all ages to stimulate the natural secretory immune systemto produce increased local tissue antibacterial immunoglobulins IgA andIgM (Del Giudice G et al. (1999) Immunol Methods 19, 148-155). Becauseof their well establish natural antimicrobial properties (Heremans J F(1970) In: Immunoglobulins, Biological Aspects and Clinical Uses, MerlerE ed. National Academy of Sciences, Washington, D.C.), secretoryimmunoglobulins can be expected to prevent or substantially reduce therisk of breast and prostate cancer. The presently disclosed methods andcompositions, directed toward prevention, promise to be applicable toreducing the risk of breast cancer in women without regard to age, race,existing risk factors, ethnic background or socio-economic status. Thisis true also of the risk of prostate cancer in men.

B Cells and Peyer's Patches.

B cells of the lamina propria secrete IgA and IgM in breast and prostatetissue. These cells originate from the Peyer's patches of the smallintestine (Owen R L (1999) Seminars Immunol 11, 157-163) and migrate tobreast and prostate after a maturation process in the circulation. Bcells from the gut enter the general circulation after stimulation byoral agents (Boyaka P N et al. (1999) Am J Trop Med Hyg 60 (4 suppl),35-45). This includes bacterial and viral challenge. The IgA and IgMproduced in breast tissue is secreted into milk (Nathavitharana K A etal. (1995) Arch Dis Chil Fetal Neonatal Ed 72, F102-F106). The IgA andIgM produced in prostate tissue is secreted into seminal fluid (Stern JE et al. (1992) J Reprod Immunol 22, 73-85). The immunoglobulins aretransported across mucosal epithelium by poly-Ig receptor mediatedtranscytosis (Mostov K E (1994) Annu Rev Immunol 12, 63-84). In allsecretions of mucosal tissues, IgA and IgM are primary antimicrobialagents. This process has been described in detail (Mestecky J and McGheeJ R (1987) Adv Immunol 40, 153-245). After identifying the types andstrains of bacteria most likely to cause breast and prostate cancer, theresearcher proposes to use inactivated forms or attenuated forms as oralchallenges to develop mucosal immunity (Viret I F et al. (1999) InfectImmunol 67, 3680-3685). As evidence of the feasibility of this concept,this same approach was used by Sabin to develop mucosal immunity againstthe poliovirus (Valtanen S et al. (2000) J Infect Dis 182, 1-5; Fiore Let al. (1997) J Virol 71, 6905-6912).

Oral Immunization.

Oral immunization can be effective for induction of specific sIgAresponses if the antigens are presented to the T and B lymphocytes andaccessory cells contained within the Peyer's patches where preferentialIgA B-cell development is initiated. The Peyer's patches contain helperT cells (TH) that mediate B-cell isotype switching directly from IgMcells to IgA B cells then migrate to the mesenteric lymph nodes andundergo differentiation, enter the thoracic duct, then the generalcirculation, and subsequently seed all of the secretory tissues of thebody, including the lamina propria of the gut and respiratory tract. IgAis then produced by the mature plasma cells, complexed withmembrane-bound secretory component, and transported onto the mucosalsurface where it is available to interact with invading pathogens. Theexistence of this common mucosal immune system explains in part thepotential of live oral vaccines and oral immunization for protectionagainst pathogenic organisms that initiate infection by firstinteracting with mucosal surfaces.

Oral Immunization is not Conventional Tumor Immunization.

In view of the foregoing examples, it can be readily appreciated that aprimary goal in the present case is not to raise conventional anti-tumorantibodies against the tumor, in contrast to existing approachescommonly used today for cancer immunotherapy. Available mucosal routesfor obtaining the desired immune response (i.e., production ofIgA/IgM/IgG1) include oral, intragastric, nasal, urogenital and rectal.Oral administration is preferred, however, because of its ease of use,whether for inducing mucosal secretion of cancer-arresting amounts ofIgA/IgM/IgG1 in contact with the gastrointestinal mucosa or at anothermucosal site. Nasal administration can be effective and convenient.

Strategies for Immunization.

A number of suitable strategies have been developed for oralimmunization, including the use of attenuated mutants, of bacteria (e.g.Salmonella) as carriers of heterologous antigens, encapsulation ofantigens into microspheres composed of poly-DL-lactide-glycolide (PGL),protein-like polymers-proteinoids, gelatin capsules, differentformulations of liposomes, adsorption onto nanoparticles, use oflipophilic immune stimulating complexes, and addition of bacterialproducts with known adjuvant properties, all of which are well known tothose of skill in the art and have been described in the literature.

Age to Begin Oral Immunization.

Prevention is one of the most important issues in cancer. It is wellknown that there is a time period, or window, during which young femalesare most susceptible to mutagenic events (e.g., ionizing radiationand/or exposure to chemical mutagens) that later predispose them tohigher than average rates of breast cancer. This window is duringpuberty (i.e. about 9 to 16 years). An oral “vaccine” will be given tovery young females (i.e. starting as early as seven years of age, orless) to induce high levels of tissue B cells that secrete protectivedimeric IgA and pentameric IgM. This same protective treatment orpreventative may also be administered to women of all ages, with thegoal is to “immunize” women against breast cancer by increasing thetissue concentrations and secretion of polymeric IgA and IgM. This verysame process can be applied to prostate and many other types ofepithelial tissues and cancers.

Rising Risk of Breast Cancer.

The risk of developing breast cancer for women in the United States hasbeen rising steadily for the past several decades. It will soon approachone in eight. We are fortunate that new treatments and more effectivescreening tests have kept mortality rates from also rising asdramatically. Nonetheless, we are still losing more than one hundredwomen per day to breast cancer in the United States alone. It isgenerally recognized by breast cancer researchers that the first line ofdefense against this disease is prevention. In the near future, thepresent know-how will continue to be used to treat these cancers as theyoccur. However, in order to improve the long-term outlook for all women,and especially if we wish our daughters to live free of this disease,major efforts must also be focused on prevention.

The Secretory Immune System and Growth Regulation as the DiscoveryOpening this New Area of Prevention.

As detailed in the preceding examples, a major breakthrough has beenmade in understanding how breast cancers grow. It was, found that in itsinitial stages, breast cancer is inhibited by the secretory immunesystem. That means this part of our immune system can stop early cancercells from growing. The well known operation of the secretory immunesystem includes, during adult life, the production by women's breasts ofmilk or milk-like fluids. Milk contains high levels of twoimmunoglobulins IgA and IgM. These are passed from mother to childduring breastfeeding. Both IgA and IgM protect the child's digestivesystem from bacterial infections. Along with protecting the child, wehave known for many years that breastfeeding lowers the risk of breastcancer. As a result of this discovery, it is now recognized that thesame immunoglobulins that protect a child from bacteria can also bemanipulated to protect the mother against breast cancer. Thisrealization also provides new insight with respect to the problem ofprevention.

Oral Immunization—Mass Applicability.

If the secretory immune system can be stimulated at times when women areknown to be most susceptible to environmental and other agents thatcause breast cancer, the occurrence of breast cancer might be preventedor at least the risk of developing this disease might be considerablyreduced. Although there have been previous studies in the literaturerelating to cancer prevention, none of the studies contemplating the useof oral immunization to treat cancer or a wide variety of infectiousdiseases, had pursued that objective beyond initial thoughts. Moreover,the application of oral immunization specifically to breast cancer hadnot received any attention. One benefit of the new oral immunizationstrategy for reducing the risk of and/or preventing breast cancer isthat oral immunization is readily adaptable to mass populations of womenof all ages and all circumstances throughout the world.

Example 40 Rat Model for Testing Oral Immunization Effects on MammaryGland Carcinogenesis

Rat Mammary Tumor Model For “Windows.”

In this Example, use of an animal model to test the effectiveness oforal immunization during specific windows of susceptibility tocarcinogens is described. This study is intended to be conducted beforeadvancing to any type of human testing. Mammary carcinogenesis in femalerodents is most effective during the developmental period that spansearly puberty through early young adulthood (FIG. 123). Singlechallenges with mammary specific carcinogens during this “window” periodcause tumors in the majority of animals within one year. Similarchallenges later during adulthood are far less effective. The results oftwo typical carcinogen experiments are shown in FIG. 123. These datasupport the conclusion that a “window” of increased susceptibilityexists during which mutations can be induced that lead to breast cancerlater in life. There is a body of evidence that indicates that this isalso true of human females. Exposure of 10 to 19 year old females toionizing radiation or chemical mutagens leads to higher than expectedbreast cancer rates later in life. Similar exposures of adult humanfemales were far less deleterious. The explanation for theseobservations is the fact that mammary gland DNA synthesis increasesduring puberty and young adulthood due to the onset of thedifferentiation program and sex hormone secretion. This programinitiates the full development of the gland. As gland terminal end buds(TEB) develop, they are the sites for mutagenesis. Clearly, DNAsynthesis is required for carcinogenesis of mammary gland. Takingadvantage of these facts, it is proposed that the secretory immunesystem can be stimulated to reduce DNA synthesis during this critical“window” and thereby diminish the risk of carcinogen induced breastcancers. In FIG. 128, it was demonstrated that IgA in the plasma offemale S-D-rats is significantly reduced at the time when carcinogenesisis most effective.

Carcinogen sensitive adolescent female rats as well as sexually maturefemales and multiparous females, both of which are more carcinogenresistant than the younger females will be studied. The rat mammarytumor is a suitable model because of the large carcinogenesis databaseavailable and the abundance of other applicable methodologies. Also,there is convincing evidence that carcinogen induced rat mammary cancersare histologically similar to those of human breast. Preferably,environmentally relevant carcinogens will be employed in the studies.While lipophilic polycyclic hydrocarbons such as7,12-dimethylbenz(a)anthracene (DMBA) and 3-methylcholanthrene (3MCA)and the soluble alkylating agent nitrosomethylurea (NMU) effectivelytransform mammary tissue with single doses, they are not found in ourenvironment. NMU is also excluded from these studies because it causesspecific changes in the ras protooncogene that are not common in humanbreast cancers. Investigators have suggested that 80 to 90% of humanbreast cancers are likely induced by environmental carcinogens.

Inhibitory compositions containing IgA, IgM and/or IgG1 will be employedto determine whether mutations leading to breast cancer occur early inlife during puberty and young adulthood, and if the control of DNAsynthesis by IgA/IgM during this critical period will attenuate theaction of carcinogens and thereby reduce the risk of breast cancer laterin life. IgA and IgM will be administered to young female animalsinitially to diminish the effects of carcinogens. These studies willthen be followed by oral “immunizations” to increase the natural levelsof immunoglobulin secreting B-cells within the mammary tissue. Thestudies will include adolescent females as well as those in mid-life. Intreated individuals, there may be some consequential delay of entry intopuberty and/or some reduction in breast development, compared tountreated individuals. This oral immunization approach is the firstattempt to deter or prevent breast cancer using the new strategy, and isfurther unprecedented by applying it early in life.

General Materials and Methods.

S-D female rats will be purchased from Harlan-Sprague-Dawley. Animalholding rooms are maintained at 23±2° C. at constant humidity on 12 hourlight/12 hour dark cycles. After anesthesia, blood will be drawn bycardiac puncture until exsanguination. The blood will be clottedovernight at 4° C. before collection of serum. The serum from individualanimals will be stored separately at −20° C. The rats will be fed anAIN-76A high fat diet which was effective in another study of mammarycarcinogenesis with S-D rats treated by gavage with the environmentallyubiquitous agents benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and2-amino-1-methyl-6-phenylmidazol[4,5-b]pyridine (PhiP). This dietsupports body weight gain at control levels even after eight weeklycarcinogen treatments. Survival rates for 41 weeks after carcinogentreatment did not differ from controls.

Rabbit polyclonal antibodies will be raised against human secretorycomponent, which will be obtained from customary commercial sources. Theantibodies will be raised and tested by Western immunoblotting withchemiluminescence detection to confirm specificity and species crossreactivity. The antibodies will be immunoaffinity purified. To measurerat IgA and IgM in serum, tissue extracts or secretion samples,radioimmunoassay (RIA) will be used with antibodies purchased fromZymed. Iodine labeling of IgA, IgM and secretory component will be doneby standard methods. A non-radioactive ELISA will also be evaluated tomeasure IgA, IgM and secretory component. The concentrations of IgA andIgM in secretions can also be estimated by Western analysis withdensitometry, according to well known procedures. Secretory componentwill be measured by RIA. RIA/ELISA data will be analyzed by computerusing logit transformations and regression analysis, as in known bythose skilled in the art.

Purified rat plasma IgA, IgM and bulk IgG will be purchased initiallyfrom Zymed. Human sIgA arid human plasma dimeric/polymeric IgA will bepurchased from Accurate Chemicals. As larger supplies become necessaryfor animal tests, plasma IgA and IgM can be purified by the preferredmethods described herein, and sIgA from colostrum. Alternatively,another purification method could be substituted, provided that ityields IgA and IgM preparations with cell growth inhibitory activitycharacteristics and purity at least equal to those described in thepresent cell growth assays.

The environmental carcinogens to be tested will be B[a]P, 1-NP and PhIP.They will be compared to a trioctanoin vehicle control. Tumors appear inresponse to B[a]P, PhIP and 1-NP at 5, 9 and 17 weeks, respectively. Thecarcinogens will be administered for eight weeks at a dose of 50μmol/rat/week. Body weight versus time will be measured. A repeatedmeasures analysis of variance (ANOVA) will be employed to determineoverall group differences in weight. Pair-wise, repeated-measuresanalysis will be employed to determine where differences occur.Cumulative mortality will be measured. The probability of survival willbe evaluated by life-table analysis with death as the end point. Thestatistical difference between pairs of groups will be evaluated by thelog-rank test. Tumor incidence will be evaluated by life-table analysiswith time of first appearance of tumor as the end point. Over theplanned duration of these experiments, the rate of spontaneous mammarytumors is not significant.

For the quantification of mammary gland development, radioisotopelabeling of DNA and estimation of numbers of tumors, applying welldescribed methods. Both right and left cervical, thoracic, abdominal andinguinal glands will be analyzed. Left glands will be fixed for wholemount estimates of the numbers of terminal end buds (TEB), terminalducts (TD) and alveolar buds (AB) structures. Carcinogenicity correlateswith the densities of TEB and TD. The effects of IgA and IgM andcarcinogens will be monitored on these structures as well as on L.I.(Labeling Index) and the numbers of tumors. The right glands will belongitudinally sectioned for autoradiography (i.e., L.I. measurements)and stained for tumor quantification. The scoring of tumors will be doneby three methods. Palpable tumors will be measured, the number of tumorsin whole mounts will be estimated by stereomicroscopy and microtumorswill be counted in the sections. The dose and timing of methyl tritiumlabeled thymidine (³H-TdR) treatment of the animals has been defined.DNA synthesis can be measured at any time prepubertal rats because theestrus cycle has not begun. After puberty, DNA synthesis is measured atestrus. Five rats will be included in each time point. This sample sizehas yielded significant (P<0.05) differences between prepubertal animalsand those at 110 days. The unpaired t test will be used to compare theresults from different age groups to determine when a significantdifference in DNA synthesis has been identified (i.e. P<0.05). The agegroups to be studied will be 30 to 35 days, 35 to 40 days, 40 to 45days, 45 to 50 days, 60 to 65 days, 80 to 85 days, 100 to 110 days, 120to 150 days, 200 to 230 days and retired breeders at 270+ days.

First, the age of young female rats will be identified in which DNAsynthesis is maximized. DNA synthesis will be monitored by ³H-TdRincorporation. This initial study is expected to confirm, under thepresent test conditions, those data reported by others in theliterature. Age groups spanning 20 days to 270+ days will be assessed.When the period of maximum DNA synthesis is identified, IgA and IgMinjections will be used to suppress DNA synthesis during this time.After the period of most rapid DNA synthesis has been identified, thefemales of that group will be treated i.p. with IgA and IgM. Todetermine dose, RIA of the serum collected from each animal group listedabove will be performed to establish the concentrations of IgA and IgMin the circulation of sexually mature adult and multiparous females.After an effective immunoglobulin dose is found, the appropriate agegroup will be treated with IgA/IgM and the effects on carcinogenesisassessed versus control animals. The doses of the immunoglobulins willbe increased until blood levels in the adolescent rat equal or exceedthose of mature females. These doses will be administered before thestart of DNA synthesis and throughout the period of carcinogentreatment. When DNA synthesis has been suppressed as judged by totallabel incorporation into DNA, measurement of L.I. and TEB measurements,the three environmental carcinogens will be administered to separategroups of fifteen rats and monitor tumor development as described above.The unpaired t test will be used to compare the results from between thecontrol group (vehicle only) and each carcinogen treated group. Thedifferences between carcinogen groups will be compared as describedabove. A significant (p<0.05) suppression of carcinogenesis and asignificant suppression of TEB development are expected to beidentified. The expected result is that carcinogens will be lesseffective in those rats receiving DNA synthesis inhibiting doses ofIgA/IgM.

Next, the conditions for inducing increased B-cell populations in breasttissue will be identified. Initially, the B-cell content of mammarytissue as a function of age will be monitored. This control study willbe correlated with the time period of maximum DNA synthesis. The contentof B-cells is expected to be low in those age groups showing a maximumDNA synthesis rate. Next, using oral challenges, the most effective“immunogen” to induce an increased population of B-cells in mammarytissue will be determined. The end point of these studies will be toinduce sufficient numbers of B-cells to prevent the “window” increase inDNA synthesis. When conditions have been established to prevent thisrise, the animal will be treated with carcinogens and monitored fortumor development and survival. The oral “immunization” is expected toreduce the effectiveness of the carcinogen.

All secretory tissues from human adults contain substantial numbers ofIgA and IgM producing immunocytes. The immunocytes in lactating humanmammary are about 0.80% IgA secreting and 10% IgM. We will use theanimal groups described above to evaluate the effect of age on IgA & IgMimmunocytes in rat mammary glands. Immunocytes in histological sectionswill be detected by fluorescence after incubation with secretorycomponent and the appropriate primary and secondary antisera. Detaileddescriptions of the fixation and detection methods have been presented.It is expected that adolescent females will have lower numbers of IgAand IgM immunocytes (p<0.05) than adults or multiparous females.Comparisons between the groups will be based on median values and theMann-Whitney non-parametric test (one tail).

Next, “oral challenges” will be used to increase the numbers of IgA andIgM immunocytes in the mammary glands of immature/pubertal female rats.In contrast to historical suggestions of oral immunization of mucosaltissues, including applications to neoplasia, the present,non-conventional “oral immunization” project preferably includes the useof immunogens that show promise with regard to breast. The mostpromising of these are non-pathogenic strains of E. coli. The first ofthese is E. coli 083 that has been used in humans to increase sIgAsecretions in colostrum. Remarkably, high levels of sIgA were induced incolostrum without causing intestinal disturbances. Ingestion by infantsor non-pregnant adults was without symptoms. The colostrum containednumerous immunocytes that secreted IgA against the O antigen of thebacteria. Furthermore, eight, or more prevalent types of E. coli inducedmilk antibodies/immunocytes against the lipopolysaccharide (LPS) of thebacteria. Indeed, even the LPS alone induced high levels of colostrumimmunocytes secreting IgA. The present study will begin with E. coli 083and the LPS from it. The methods of analysis of antibodies in the bloodand in rat colostrum will be done as described. Dosing of the bacteriumand LPS will be developed to block the “window” of DNA synthesis. Wheneffective dosing regiments have been found, we will analyze the effectsof the carcinogens to determine if they are effective when DNA synthesisis suppressed. Also, the state of differentiation of the gland will beanalyzed by measuring terminal end buds (TEB), terminal ducts (TD) andalveolar buds (AB). Both carcinogenesis and differentiation are expectedto be inhibited.

In a third phase of the studies, it will be determined if disruption ofthe function of the secretory immune system causes adult and multiparousfemale rats to become more sensitive to carcinogens. Virgin female ratsof 114 days or older will be studied as will retired breeders of morethan 250 days age. These animals will be treated with antibody againstthe poly-Ig receptor. The doses of antiserum to disrupt the secretoryimmune system will be established by monitoring IgA/IgM secretion intobile, uterine fluids and breast milk. Also, mammary DNA synthesis willbe monitored. When secretion is blocked effectively, the susceptibilityof these animals to carcinogens will be measured. The disruption of theinteraction of IgA/IgM with the poly-Ig receptor is expected to increaseDNA synthesis in the mammary gland and therefore increase susceptibilityto carcinogens.

Because rats do not undergo menopause, a different approach toinvestigating the possible “window” in mid-life females will be used.For this study, the interaction of IgA and IgM with the poly-Ig receptorwill be disrupted using polyclonal antibodies against the receptor. Theantibodies will be confirmed effective by blocking ¹²⁵I-IgA binding tobreast cancer cell receptors using methods. The effect of theseantibodies in vivo will be measured by monitoring DNA synthesis in theadults of 110 to 120 days, 200 to 220 days and retired breeders. Also,the secretion of IgA, IgM, secretory component and J chain into bile,uterine fluids and breast milk will be monitored by the methodsdescribed. Similar methods with J chain polyclonal antibodies haveproven very effective in rats. When the secretions have been diminishedsatisfactorily, antibody treated animals will be treated simultaneouslywith carcinogens. It is expected that DNA synthesis will increase inadult and multiparous animals treated with the antibodies and that thecarcinogens will become more effective.

Applicability to Humans.

Human female breast cancer incidence rates increase dramatically afterage 50 and now approach one in eight by age 75. The existing datasuggest that the causal mutations most likely occur at earlier ages.However, milk/breast secretions decrease dramatically after menopause.Perimenopausal and postmenopausal women may also have a previouslyunrecognized “window” of increased vulnerability because the activity ofthe secretory immune system decreases with the approach of mid-life.Accordingly, the IgA, IgM and IgG1 inhibitor compositions will also beemployed to aid in determining whether mutations can arise later in lifedue to the natural age related reduction in the growth inhibitoryfunction of the secretory immune system.

Example 41 Bacterial Oncogenesis and Prevention by Oral Immunization

The present example addresses the cause of breast and prostate cancer,as well as cancers of other steroid hormone responsive tissues, from theperspective of determining what is causing the normal mucosal epithelialcells of these tissues to become transformed to the malignant state. Itis now proposed that certain bacteria are carcinogenic (oncogenic),especially in mucosal epithelial tissues, and a screening procedure forisolating and identifying oncogenic bacteria, or bacterial that arelikely to be oncogenic has been devised.

Also presented herein is a two-fold immunization plan to prevent orreduce the risk of occurrence of cancer of the breast, prostate, andother steroid hormone responsive mucosal endothelial tissues. The firstkind of immunization involves immunizing an individual in theconventional way to invoke a natural immune response in whichantibacterial immunoglobulins target and eliminate specific oncogenicbacteria. The second kind of “immunization,” which was previouslyunknown, is to stimulate the natural secretory immune system to producesteroid hormone reversible cell growth inhibitors (i.e., “immunoglobulininhibitors”), which the inventor has discovered are active forms of IgA,IgM and IgG1. These inhibitors have activity for regulating steroidhormone reversible cell growth in mucosal epithelial tissues, such asbreast and prostate. Alternatively, the individual may be “passivelyimmunized” by local or systemic administration of IgA, IgM and IgG1. Bymeans of their cell growth regulatory function, the active forms of IgA,IgM and IgG1 are believed by the inventor to protect the mucosalepithelial tissues from the deleterious effects of bacterial oncogenesiswhich lead to cancerous cell growth.

As disclosed hereinabove and in U.S. patent application Ser. No.09/852,958 PCT/US2001/15183 entitled “Compositions and Methods forDemonstrating Secretory Immune System Regulation of Steroid HormoneResponsive Cancer Cell Growth,” hereby incorporated herein by reference,the secretory immune system immunoglobulins IgA, IgM and IgG1 are potentinhibitors of steroid hormone responsive cancer cell growth inchemically defined serum-free Medium. This serum-free cell culturesystem constitutes a preferred in vitro model of in vivo tumor cellgrowth that is superior to previously available serum-free systems. Theinhibitory activity is mediated by poly-Ig receptor or a poly-Ig-likereceptor. Among other things, this discovery has strong physiologicalsignificance in humans related to the well-known production of IgA, IgMand IgG1 in breast tissue and the secretion of these sameimmunoglobulins into breast milk. In the past, the IgA, IgM and IgG1 ofmilk were thought to serve only as an antibacterial protection for thesuckling offspring. These same immunoglobulins, particularly in the formof polymeric IgA and pentameric IgM and IgG1, may also protect themother and provide a new means of preventing or reducing her risk ofbreast cancer. Similar negative regulation by IgA, IgM and IgG1 has alsobeen demonstrated by the inventor in androgen responsive prostate cancercells. Analogous results are also indicated in steroid hormoneresponsive cancers of all other mucosal epithelial tissues that eithersecrete or are bathed by IgA, IgM and IgG1 in the body. These includenot only tissues of the breast, prostate, pituitary and kidney, but alsoany other tissue that lines a cavity or secretes IgA/IgM/IgG1, such astissues of the gastrointestinal tract (i.e. oral cavity mucosa,salivary/parotid glands, esophagus, stomach, small intestine and colon),tear ducts and nasal passages, liver and bile ducts, bladder, pancreas,adrenals, kidney tubules and glomeruli, lungs, the female reproductivetract (i.e. ovaries, fallopian tubes, uterus, cervix and vagina) and thesecretory anterior pituitary gland. All of these glandular/mucosaltissues either secrete or are bathed by polymeric IgA, secretory IgA(sIgA), IgM and IgG1. Cancers arising from these tissues account for 80%of the epithelial malignancies of humans.

In light of the discovery that the secretory immune systemimmunoglobulins IgA, IgM (and IgG1 in humans) are potent inhibitors ofsteroid hormone responsive cancer cell growth, in the preceding.Example, it has been demonstrated how the steroid hormone responsivetissues in the body may be protected from the cancer causing actions ofcertain environmental carcinogens by enhancement of the IgA, IgM andIgG1 secreted by or coming in contact with those tissues. In this way,DNA synthesis dependent mutations can be prevented or substantiallyreduced in those tissues.

Certain bacterial products, either alone or in cooperation withleukocytes, are responsible for production of “reactive oxygen andnitrogen” that lead to malignant transformation of breast and prostateepithelial cells. Immunity to these oncogenic bacteria can conferresistance to this process and thereby reduce the risk of breast andprostate cancer. By employing the bacterial screening procedures thatare described below, bacteria that are likely to be inducers of cancerin vivo are identified. These bacteria, or a combination of bacteria, orimmunogens derived from the oncogenic bacteria, can then be used todevelop specific antimicrobial therapies. One such antimicrobial therapyincludes the production of secretory immunity via oral administration ofthe inactivated or otherwise attenuated bacteria to confer mucosalimmunity. Alternatively, nasal or rectal administration routes may beemployed to produce mucosal immunity in an individual considered to beat risk of developing cancer in a mucosal tissue. Another means ofprotecting an individual against the oncogenic action of the bacteriaisolated or identified as set forth below is to induce systemic immunityto the bacteria, using conventional techniques for raising systemicantibodies to a microorganism.

Moreover, by employing conventional diagnostic immunology and otherimmune-based tests of plasma or other bodily secretions, it can bedetermined if an individual has been or is actively infected by thesuspected oncogenic bacteria, which has been isolated or identifiedaccording to the screening procedures described below. With thisinformation, predictions can be made as to which individuals may be athigher risk for development of cancer in the affected tissue.Alternatively, or additionally, a variety of conventionalmetabolic/chemical inhibitor approaches may be employed to destroy thepotentially oncogenic bacteria in the affected tissues. For example,administration of an effective dose of an appropriate antibiotic to anindividual infected by an oncogenic bacteria.

In light of the discovery regarding the role of the natural secretoryimmune system in regulating the growth of cancer, and finding indirectsupport in the literature, it is now concluded that bacterial infectionsare likely to be an important factor in development of prostate cancer,and that bacteria are also likely to be a primary cause of cancers ofbreast and other mucosal epithelial tissues. Accordingly, the followingscreening procedures are provided for isolating and identifying bacteriafrom breast and prostate sources and assessing their transformingactivity.

Screening Procedure for Identifying Carcinogenic Bacteria.

Human milk will be collected from pregnant volunteers either directly orvia professional organizations working with nursing mothers.Alternatively, nipple aspirate fluid will be obtained from non-cancerousvolunteers and breast cancer patients prior to surgery or chemotherapy,preferably as described (Trock B and McLesky S Proceedings of the Era ofHope 2000 Meeting, Department of Defense Breast Cancer Research Program,Atlanta, Ga., June, 2000). Breast tissue samples from non-surgicalvolunteers will be collected under conditions that exclude skin originbacteria. Breast cancer samples obtained during surgery will also bedirectly cultured and evaluated. Those samples will include normalbreast tissue (e.g. from reduction mammoplasty) and tumor specimens frombreast cancer patients.

Specimens of semen/seminal fluid will be obtained from normal volunteersof different ages. Because cancer causing mutations may be present forseveral years before the clinical manifestation of disease, samples willbe collected from young adult men <35 years of age as well as from meninto their seventies (highest rate). In addition, surgical samples willbe cultivated and otherwise analyzed to identify the types of bacteriapresent and their relative frequencies. The samples will be classifiedas (i) bacterial prostatitis, (ii) nonbacterial prostatitis, and (iii)asymptomatic inflammatory prostatitis (Lipsky B A (1999) Am J Med 106,327-334).

Special care will be given to the analysis of clinical samples forbacterial content. Some considerations have been discussed by others(Sandin R L and Rinaldi M (1996) Infect Dis Clin North Am 10, 413-430).Precautions will be taken to avoid inclusion of extraneous bacteria inthe samples, and to ensure quality control, including those indicated.Gram stain negative versus gram stain positive bacteria will beclassified. Gram stain negative bacilli cause most prostatitis (Lipsky BA (1999) Am J Med 106, 327-334). For breast samples, this must still beestablished. This is the first selection process to be used to reducethe number of possible bacteria.

The next selection process will use colony derive bacteria to conductthe “Ames Test” to identify bacteria producing mutagens (Ames B N (1979)Science 204, 587-593). This test is based on the scientifically acceptedconcept that DNA damage appears to be the major cause of cancer. Thisassay employs an in vitro mutagenesis test using the bacteriumSalmonella. The culture medium from each form of bacteria isolated canbe tested directly for mutagenic activity using any of several strainsof Salmonella developed for this purpose. The different types ofscreening methods have been reviewed (Hill D C (1998) Adv Biochem EngBiotechnol 59, 73-121). Addition improvements in the Ames Test have beenintroduced to provide more quantitative evidence that the assay isproviding significant results with respect to cancer bioassays (Bogen KT (1995) Environ Mol Mutagen 25, 37-49). The results will be analyzed bystatistical methods (Kim B S and Margolin B H (1999) Mutat Res 436,113-122). The results of this test will establish which bacterialisolates produce mutagenic metabolites (e.g. reactive oxygen andnitrogen species).

The Ames Test can also be applied to demonstrate that the bacteria causean “oxidative burst” mediated by neurophils and macrophages. In thiscase, the leukocytes are incubated with the bacteria to generate theactive mutagenic species. This approach resolves the issue of whetherthe products of the bacteria are the mutagens themselves or if theactivation of leukocytes is required. The various kinds of mutagenesiswill be considered in light of human oncogenesis criteria (Miller J H(1996) Cancer Surv 28, 141-153).

Another type of selection has special application to breast cancer. Milkcontains the protein lactoferrin (Masson D and Taylor C (1978) J ClinPath 31, 316-327). It is well known to be bactericidal by virtue of itshigh affinity for iron (FeIII) (Arnold R et al. (1977) Science 197,263-265). Most bacteria have an absolute requirement for FeIII to grow.However, some have developed “lactoferrin receptors” that permit them toacquire the necessary iron even through it is in complex withlactoferrin. The inventor predicts that mutagenic types of bacteria inbreast secretions/milk will survive and grow in the presence of highconcentrations of lactoferrin. This offers a potent means of selectingfor the bacteria being sought.

Bacteria that meet the criteria described above will be cultured and themedium tested with non-tumorigenic human breast epithelial cells todetermine if the cells are altered to a malignant phenotype. The test ofaltered growth will first be done in serum-free chemically definedmedium, prepared as described the foregoing examples and in U.S. patentapplication Ser. No. 09/852,958 PCT/US2001/15183 entitled “Compositionsand Methods for Demonstrating Secretory Immune System Regulation ofSteroid Hormone Responsive Cancer Cell Growth”, or in Moreno-Cuevas andSirbasku et al. (2000b), the disclosures of which are incorporatedherein by reference. Transformed cells have reduced growth factor andadhesion requirements. Also, the cells will be tested for colonyformation in standard assays. Normal epithelial cells will not formcolonies in soft agar. Tumor or transformed cells will form colonies.There is a very strong correlation between colony forming activity insoft agar and tumorgenicity in host animals. These tests are expected toconfirm that the mutagenic effects seen with the Ames Test can betranslated to transformation of human breast cancer cells. Also, normalhuman prostate epithelial cells are available and will be used toperform a similar sequence of studies.

In addition to meeting the foregoing applicable selection criteria, someof the bacteria are expected by the inventor to also possess animmunoglobulin protease activity, i.e., its own “immunoprotective”mechanism. Both seminal fluid and breast secretions contain highconcentrations of IgA. IgA is secreted by prostate and breast epithelialcells. The secreted IgA acts to kill bacteria in these fluids therebyprotecting the tissue. Several types of organisms are known to secreteproteases that cleave the IgA into inactive Fab and Fc components.Examples are Streptococcus pneumoniae (Wani J H et al. (1996) InfectImmun 64, 3967-3974), Haemophilus influenza serotype b (Poulsen K et al.(1989) Infect Immun 57, 3097-3105), Neisseria gonorrhoeae (Simpson D Aet al. (1988) J Bacteriol 170, 1866-1873), Bacteroides melaminogenicus(Mortensen S B and Kilian M (1984) Infect Immun 45, 550-557). These areonly a few examples of bacterial protease activities that have beendescribed in the literature and which are consistent with, and provideindirect support for, this oncogenic bacterial selection criterion.

Finally, to define the bacteria identity, the inventor will apply PCRmethods (Wagar E A (1996) J Clin Lab Anal 10, 331-334). Techniques thatmay be applied include, for example, (1) use of specific PCR primers forknown and new bacteria, (2) PCR amplification of conserved 16S rRNAsequences, and (3) RDA-PCR which is also called “reverse PCR”. Thistechnique can be used to identify unique infectious agents in diseasetissues. Additional PCR technology is available for most of the microbesthat are likely to be encountered.

Although the foregoing protocol has been described with respect tobreast and prostate fluids and tissue specimens, it should be understoodthat similar protocols can be employed with fluids, secretions or tissuespecimens from other mucosal epithelial tissues, including those of thegastrointestinal tract (i.e. oral cavity mucosa, salivary/parotidglands, esophagus, stomach, small intestine and colon), tear ducts andnasal passages, liver and bile ducts, bladder, pancreas, adrenals,kidney tubules and glomeruli, lungs, the female reproductive tract (i.e.ovaries, fallopian tubes, uterus, cervix and vagina) and the secretoryanterior pituitary gland.

Reduction of Breast Cancer Risk by Immunization.

One very important application of the bacteria that are identified asoncogenic, or likely to cause cancer in breast tissue, is to use oralchallenges to develop mucosal immunity against the bacteria. For thepurposes of this disclosure, the term “oncogenic bacteria” refers to theforms of bacteria that cause cancer. According to a preferred regime forpreventing or reducing the risk of breast cancer, oral immunization willbe administered to men and women of all ages to stimulate the naturalsecretory immune system to produce increased local tissue antibacterialimmunoglobulins IgA and IgM. Existing techniques will be employed, suchas those described (Del Giudice G et al. (1999) Methods 19, 148-155).Because of their established natural antimicrobial properties (HeremansJ F (1970) In: Immunoglobulins, Biological Aspects and Clinical Uses,Merler E, ed. National Academy of Sciences, Washington, D.C., 1970), theinventor expects that the secretory immunoglobulins will prevent orsubstantially reduce the risk of breast and prostate cancer by targetingand eliminating the bacteria. The first phase of this disclosure (i.e.identification of oncogenic bacteria) is a first step toward achievementof the second phase, i.e., implementing natural immune system preventionmethods. Such a prevention method is applicable to reducing the risk ofbreast cancer in women without regard to age, race, existing riskfactors, ethnic background or socio-economic status. Similarly, butpreferably using oncogenic bacteria identified in prostate tissue, thenatural secretory immune system will be stimulated to protect againstprostate cancer in men. In a preferred embodiments, the naturalsecretory immune system will be stimulated to eliminate the oncogenicbacteria via conventional antigen-antibody recognition chemistry, and/orto protect breast and prostate tissue from the deleterious effects ofbacterial oncogenesis via the non-conventional cell growth inhibitoryeffects of the secretory immune system.

B cells of the lamina propria of breast and prostate tissue secrete IgAand IgM. These cells originate from the Peyer's patches of the smallintestine (Owen R L (1999) Seminar Immunol 11, 157-163) and migrate tobreast and prostate after a maturation process in the circulation. Entryof B cells into the circulation is stimulated by oral agents (Boyaka P Net al. (1999) Am J Trop Med Hyg 60 (4 suppl), 35-45). This includesbacterial challenge. The IgA and IgM produced in breast tissue isdestined for secretion into milk (Nathavitharana K A et al. (1995) ArchDis Chil Fetal Neonatal Ed 72, F102-F106). The IgA and IgM produced inprostate tissue are destined for secretion into seminal fluid (Stem J Eet al. (1992) J Reprod Immunol 22, 73-85). The immunoglobulins aretransported across mucosal epithelium by poly-Ig receptor mediatedtranscytosis (Mostov K E (1994) Annu Rev Immunol 12, 63-84). In allsecretions of mucosal tissues, IgA and IgM are primary antimicrobialagents. This process has been described in detail (Mestecky J and McGheeJ R (1987) Adv Immunol 40, 153-245).

After identifying the types and strains of bacteria most likely to causebreast and prostate cancer, inactivated forms or attenuated forms of thebacteria will be used as oral challenges to develop mucosal immunityusing known techniques such as those described (Viret J F et al. (1999)Infect Immun 67, 3680-3685). A similar approach was used by Sabin todevelop mucosal immunity against the poliovirus (Valtanen S et al.(2000) J Infect Dis 182, 1-5; Fiore L et al. (1997) J Virol 71,6905-6912).

Although oral immunization against breast cancer has been describedabove, it should be understood that protection against cancers of theprostate or other mucosal epithelial tissues, including those of thegastrointestinal tract (i.e. oral cavity mucosa, salivary/parotidglands, esophagus, stomach, small intestine and colon), tear ducts andnasal passages, liver and bile ducts, bladder, pancreas, adrenals,kidney tubules and glomeruli, lungs, the female reproductive tract (i.e.ovaries, fallopian tubes, uterus, cervix and vagina) and the secretoryanterior pituitary gland, may be achieved similarly.

In addition to inducing a conventional type of mucosal immunity againstthe oncogenic bacteria, a second kind of “immunization,” will also beemployed in which the natural secretory immune system is stimulated toproduce active forms of IgA, IgM and IgG1 that have activity forregulating cancer cell growth in mucosal epithelial tissues, especiallythe steroid hormone responsive tissues of breast and prostate.Alternatively, an individual may be “passively immunized” by local orsystemic administration of IgA, IgM and IgG1 to inhibit cancer cellgrowth. As a result of their cell growth regulatory function, the activeforms of IgA, IgM and IgG1 are expected to protect those types oftissues from the deleterious effects of bacterial oncogenesis that leadto cancerous cell growth. For example, by reducing the “imprinting” ofcancer related genetic changes in prepubescent females or by preventinggrowth of early stage tumors in postmenopausal women. Preferably theIgA, IgM and IgG1 are raised against specific oncogenic bacteria,however a broad spectrum of IgA, IgM and IgG1 molecular species appearto exert steroid hormone reversible growth inhibition in these tissues.Continuing studies are directed at pinpointing the most effectivespecies of IgA, IgM and IgG1 for inhibiting cancer cell growth arisingfrom a given tissue and suppressing the effects of transformation.

Conclusions.

A bacterial origin for certain cancers is consistent with, andindirectly supported by the work of others relating to the possibleinvolvement of bacteria with Hodgkin's disease. Because manyreproductive, child bearing and socio-economic patterns are shared asrisk factors for breast cancer and Hodgkin's disease in young women,there may well be a common etiology. Moreover, in light of the fact thatno viral origin of human breast cancer has been established to date, theinventor concludes that other more common infective agents are morelikely the cause of breast and prostate cancer. One published study hasused Cytomegalovirus (CMV) infection distribution as a surrogate to testthe hypothesis that breast cancer may be of infectious origin(Richardson A (1997) Med Hypotheses 48, 491-497). Although it is notlikely that CMV is causative for breast cancer, it is found in humanmilk and is transmitted to offspring during the breast-feeding period(Diosi P (1997) Roum Arch Microbiol Immunol 56, 165-178). Those reports,viewed in light of the present disclosure, support the inventor'salternate interpretation of that information, i.e., that fortuitousbacterial infection, which spreads like viral infections, is actuallythe origin of breast cancer.

Human milk contains many microorganisms/bacteria. To date, none havebeen identified that are primary candidates as causative agents ofbreast cancer. The published work pertaining to milk microbiology willbe of great benefit when reevaluated by the appropriate discriminatorsof the present screening procedure for identifying oncogenic bacteria.The existing literature contains many candidates that will be examinedas primary causative agents for breast cancer, employing the screeningprocess described herein.

The “reactive outbursts” from bacterial-challenged leukocytes may serveas an additional cause of cancers of the male reproductive tract,including those of prostate. Although this proposal has not beenpresented before regarding a mechanism for the development of prostatecancers, it is consistent with the results of studies reported in theliterature that neutrophil and macrophage overproduction of reactiveoxygen species damage the tissues and sperm (Ochsendorf F R (1999) HumanReprod Update 5, 399-420). Because prostate cancer increasesdramatically with age, one focus of the inventor's furtherinvestigations will be on microorganisms that are common to men over 35years of age. A previous study by others has shown that theEnterobacteriaceae are more frequently involved in prostatitis in thisage group than in younger men (Joly-Guillou M L and Lasry S (1999) Drugs57, 743-750). The Enterobacteriaceae, as well as other potentiallyoncogenic bacteria, will be examined in ongoing studies as primarycausative agents for breast cancer, employing the new screening process.

The secretory immune system is an integral part of the physiology of allmucosal epithelial tissues. Most mucosal tissues secrete immunoglobulins(IgA and IgM) into the lumen of biological passageways. Although breast,prostate, pituitary and kidney cancer cells were employed in theforegoing examples, it should be understood that any tissue that lines acavity and/or secretes IgA/IgM is a candidate for the same or similarcompositions and methods for the diagnosis, treatment, deterrence orprevention. These include the gastrointestinal tract (i.e. oral cavitymucosa, salivary/parotid glands, esophagus, stomach, small intestine andcolon), tear ducts and nasal passages, liver and bile ducts, bladder,pancreas, adrenals, kidney tubules and glomeruli, lungs, the femalereproductive tract (i.e. ovaries, fallopian tubes, uterus, cervix andvagina) and the secretory anterior pituitary gland. All of theseglandular/mucosal tissues either secrete or are bathed by polymeric IgA,secretory IgA (sIgA) and IgM. Cancers arising from these tissues accountfor 80% of the epithelial malignancies of humans.

As discussed in Example 37, it is interesting to note that in ataxiatelangectasia (A-T) there is an increased incidence of malignancies,with epithelial carcinomas being the predominate kind. Laboratoryevaluations of A-T patients also show, among other abnormalities thatabout 75% of the patients are deficient in IgA and IgM. A number ofstudies have indicated that female relatives of A-T patients sufferexcess risk of breast cancer (Easton D F (1994) Int. J. Radiat. Biol 66(6 Suppl), S177-S182) or gastric cancer (J. O. Bay et al. (1998) Int. J.Oncol. 12, 1385-1390). The contribution of heterozygous A-T mutations tofamilial breast cancer is believed not to significant (Chen J et al.(1998) Cancer Res. 58, 1376-1379).

Prior to the present invention, the ability to arrest cell proliferationof early, steroid hormone responsive mucosallepithelial malignancies hasnever been attributed to IgA/IgM/IgG1. In addition to looking at certainbacteria as potential causes of malignancy, exposure to non-pathogenicbacteria may serve to continuously stimulate the body's production ofprotective levels of IgA/IgM/IgG1 to protect against, or counteract, thecell proliferation-causing effects of the harmful bacteria.

Example 42 Treatment of Steroid Hormone Responsive Breast or ProstateCancer by Administration of IgA/IgM/IgG1

In this example it is demonstrated that prolonged inhibition of cancercell growth by IgA/IgM causes cell death. This effect is exploited intherapeutic methods that have been devised.

In the in vitro assays described in preceding Examples, which areconsidered to be model systems for predicting in vivo tumor growtheffects, IgA and IgM were shown to behave as steroid hormone reversibleinhibitors of ER⁺ breast and AR⁺ prostate cancer cell growth in theclassical sense of the long sought after chalones. During the initialstages, the immunoglobulins arrest cell growth without causing celldeath. Steroid hormones can reverse the inhibition during this period.However, as with most cancer cells, prolonged blockage of the cell cyclecauses cell death (this is the well known basis for chemotherapy).Accordingly, these in vitro studies are the basis for the in vivotherapeutic use of IgM in rats to block the growth of carcinogen-inducedmammary tumors and to treat existing tumors after induction, or thosearising from implantation of MTW9/PL2 cells. In contrast to classicalchemotherapy that attacks cells of many different types, the effects ofIgA and IgM are specific for mucous epithelial cells and are non-toxicto normal organs.

Preferably the most active forms and/or subtypes of IgA/IgM/IgG1 will beemployed, i.e., those forms of immunoglobulins that act as negativegrowth regulators for human breast and prostate cancer cells. Thesubclasses and pertinent allotypes of IgA will be investigated forgrowth regulating activity with human breast and prostate cancer cellsin serum-free defined culture and for specific binding of ¹²⁵I-labeledimmunoglobulin to cell membrane receptors. The presently disclosedresults strongly imply that polymeric forms are the primary or onlybiologically active immunoglobulins. To establish this conclusively, theeffects of monomeric, dimeric and polymeric IgA on the growth of the ER⁺human breast cancer cell lines T47D, MCF-7 and ZR-75-1 will be assessed,employing the above-described growth assay procedures and materials.Cell numbers will be determined with triplicate dishes and the resultsconverted to cell population doublings (CPD) to allow a directcomparison of the specific activities of each IgA form. Each form of IgAor fragment will be ¹²⁵I-labeled by the chloramine T method. The labeledforms will be used to assess specific binding as total binding minusbinding in the presence of a 100-fold excess of the same unlabeledpreparation. For each fragment or protein (i.e. those that mediateestrogen effects), the time, concentration and temperature dependence ofbinding will be assessed. Scatchard analysis will be used to estimatethe numbers of sites per cell and association constants (Ka). Reciprocalcompetitions with unlabeled and labeled dimeric IgA will be used toconfirm that the purified types or fragments associate with the samereceptors.

The IgA1 and IgA2 will be purified from serum and human colostrum asdescribed. The monomeric, dimeric and polymeric forms of each will bepurified by size exclusion and ion exchange FPLC. If IgA2 only possessactivity, it will be further separated into the A2(m)1 and A2(m)₂allotypes. The purifications will be monitored exactly as described Ithe literature. If the most active form is dimeric (and polymeric), itwill be additional strong evidence that the poly-Ig receptor ismediating the growth response. Were the monomers found to havesignificant activity, that will imply that the poly-Ig receptor may notbe the (only) active mediating site. Next, the active IgA will befragmented beginning with IgA protease that cleaves at the classicalhinges. The methods will yield separable Fab fragments from IgA1 as wellas a larger fragment containing the J chain, the secretory component andthe Fc fragments, using standard techniques that are known to those ofskill in the art and which can be readily implemented. Each specifiedfragment will be purified and assayed for growth mediating effects andreceptor binding.

Following the confirmatory studies, preferably the most activeimmunoglobulin species (e.g., dimeric IgA, polymeric IgM, and/or IgG1)will be administered to other animal subjects or human patientssuffering from a hormone responsive breast or prostate cancer, or otherglandular/mucosal epithelial cancer. Preferably an effective dosage ofthe immunoglobulin composition will be introduced directly as one ormore intravenous treatments using known methodologies for optimizingdosage and delivering it to the subject. Administration can be doneintraperitoneally or subcutaneously as well. It should not be overlookedthat this treatment protocol is quite different from conventionalimmunotherapies that rely entirely on effecting passive immunity todisease organisms and/or their antigenic determinants. In the presentcase, the treatment is designed to primarily provide the necessary leveland/or forms or subtypes of polymeric/dimeric IgAs, pentameric IgMand/or IgG1 s for binding with the respective poly-Ig and Fcγ receptorson the target cells sufficient to produce the desired inhibition orarrest of cell proliferation.

Formulations and Processes.

For introduction into the body, pharmaceutical compositions containingthe immunoglobulin inhibitors are manufactured in a manner that is wellknown in the art, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping, and lyophilizing processes. The physiologically acceptablecarriers are non-toxic to recipients at the dosages and concentrationsemployed. The formulation used varies according to the route ofadministration selected (e.g., solution, emulsion, capsule). Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Pharmaceutical formulations suitable forparenteral administration may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiologically buffered saline. See,generally, “Remington's Pharmaceutical Science,” 16th Edition, Mack, Ed.(1980). For inhalation, the immunoglobulin inhibitors can be solubilizedand loaded into a suitable dispenser for administration (e.g., anatomizer, nebulizer or pressurized aerosol dispenser).

An “effective amount,” as used herein, is defined as that quantity whichalleviates, to any degree, or eliminates the condition for which themammal is being treated. The determination of an effective dose is wellwithin the capability of those skilled in the art. For any composition,the therapeutically effective dose can be estimated initially either incell culture assays (e.g., one of the model assay systems describedherein), or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

The immunoglobulin inhibitors can be administered individually, togetheror in combination with other drugs or agents. For example, ananti-cancer composition is prepared by conjugating a cytotoxic agent ora chemotherapeutic agent to an immunoglobulin inhibitor of steroidhormone responsive cancer cell growth, the inhibitor comprising IgA, IgMor IgG1, or any combination of those.

Example 43 Monoclonal Antibodies that Mimic or Block IgA or IgM Bindingto the Poly-Ig Receptor

In this Example, the use of new monoclonal antibodies that act like IgAand IgM to inhibit breast and prostate cancer growth by binding to thepoly Ig receptor, or that act to block the binding of IgA/IgM to thepoly-Ig receptor is described. Also described is a method of developinghybridoma cells that secrete both mimicking and blocking antibodies. Themonoclonal antibodies will be raised as described (Kohler G and MilsteinC (1975) Nature (Lond) 256, 495).

Mimicking Antibodies.

IgA and IgM mimicking monoclonal antibodies will be used in treatmentprotocols either alone or with conventional anti-hormone therapy. Theyalso will be used in diagnostic methods to analyze patient specimens forpoly-Ig receptor content by immunohistochemistry and other standardimmunological techniques that are well known in the art.

Blocking Antibodies.

A second class of monoclonal antibody secreting hybridoma cells will beobtained from the same protocols used to generate the hybridoma cellsthat secrete mimicking antibodies. This second type of blockingmonoclonal antibodies prevents the binding of IgA to the poly-Igreceptor. The hybridoma cells that secrete this type of antibody and theantibodies themselves are useful reagents. Blocking antibodies will havea variety of therapeutic and/or diagnostic uses.

Features.

One advantage of using monoclonal antibodies that mimic the IgA/IgMbinding to the poly-Ig receptor is that the poly-Ig receptor can betargeted as a new site for anticancer intervention. While commerciallyprepared polyclonal antibodies against the receptor are available(Accurate Chemicals), there are no reports of their applicability tohuman therapy. It is not likely that rabbit polyclonal antibodiesagainst the receptor will be useful in humans due to the strongantigenic response they will elicit. Also, there are two reports ofpanels of monoclonal antibodies directed against epitopes of IgA (ReimerC B et al. (1989) Immunol Lett 21, 209-216) and IgA plus the receptor(Mestecky J et al. (1996) J Immunol Methods 193, 103-148). None of thesemonoclonal antibodies has been tested for activity as an anticanceragent, nor is there any evidence that any act on the poly-Ig receptor toeither mimic or block the action of IgA on breast or prostate cancercells or on cancer cells of any of the other IgA or IgM secretingtissues of the body. One monoclonal antibody, MAB 6G11, has beendescribed that binds to domain 1 of the poly-Ig receptor (Bakos M A etal. (1994) Molecular Immunology 31, 165-168). This same domain alsobinds IgA and IgM, implying that MAB 6G11 may be a blocking or possiblya mimicking antibody. However, direct studies of this aspect were notreported, and the monoclonal antibody was not used for anti-cancerpurposes.

Examples of Other Types of Monoclonal Antibodies to Receptors.

There have been similar projects based on the receptors for otherhormones and growth factors. The use of blocking monoclonal antibodiesas therapy for cancer is known (Baselga J and Mendelsohn J (1994)Pharmacology Therapeutics 64, 127-154). The best example is themonoclonal antibodies raised against the epidermal growth factor (EGF)receptor Baselga J and Mendelsohn J (1994) Breast Cancer Res Treat 29,127-138). Another example is the monoclonal antibody rhuMab against theHER2/Neu proto-oncogene receptor which is over expressed by breastcancer cells Baselga J et al. (1996) J Clin Oncol 14, 737-744). Theseimmunoglobulins are designed to block the growth stimulating effects ofEGF/transforming growth factor (TGFα). Both EGF and TGFα cause cancercell growth including breast and prostate. Anti-EGF and anti-HER2/neureceptor monoclonal antibodies are now commercial anticancer products.

The Target is a Negative Acting Receptor.

In the case of growth factor receptor directed antibodies, the growthfactor competes for (and often neutralizes) the inhibiting action of theimmunoglobulin, which can be a significant problem. In contrast, thepresently described mimicking antibodies target a negative actingreceptor. The presence of endogenous IgA or IgM has no effect becausethe monoclonal antibody and the natural ligand have the same function,i.e., they both inhibit growth. There is no need to be concerned aboutthe presence of IgA or IgM, as they will not interfere with thistreatment. The mouse monoclonal antibodies against the poly-Ig receptorwill be converted to human immunoglobulins by genetic engineering. Thiswill prevent an immunological response against the mouse epitopes thatwill reduce antibody effectiveness. Monoclonal antibody therapy isnon-invasive and can be administered frequently over a long duration.Both mimicking and blocking monoclonal antibodies are important becauseboth are expected to have therapeutic value. The poly-Ig receptor islocalized in mucosal tissues (e.g. GI tract, lungs, breast ducts,prostate gland, uterine lining, ovary, kidney tubules and urinary tract,and salivary gland) (Brandtzaeg P (1995) Acta Path Microbiol ImmunolScand 103, 1-19). An important advantage of this disclosure is thatmonoclonal antibodies against the breast/prostate poly-Ig receptor canalso be expected to have therapeutic effects with cancers of at leastsome of these other tissues.

Development Protocols.

Various strategies may be used to raise monoclonal antibodies to thehuman poly-Ig receptor. One approach is to use standard solid-phasechemical synthesis to prepare peptides corresponding to the known aminoacid sequence of the extracellular domain of the poly-Ig receptor. Theextracellular ligand-binding domain, which is approximately 80% of thewhole receptor, was first named the “secretory component” because it wasfound in association with secreted IgA and IgM. Monoclonal antibodiesagainst secretory component can be assayed to determine if they act asmimicking or blocking agents, employing a cell growth assay describedherein. An alternative approach will be to use a combination ofimmunoprecipitation, affinity chromatography and immunoaffinitychromatography to purify the intact (complete) poly-Ig receptor. Thepurified receptor will then be used to raise monoclonal antibodies,which can then be screened for mimicking and blocking activity in asuitable cell growth assay described above.

Example 44 Delivery of Chemotherapeutic Agents and Cytotoxins to CancerCells via IgA/IgM/IgG1 or Monoclonal Antibodies to Poly-Ig Receptor

In this Example, polymeric IgA/IgM and monoclonal antibodies to thepoly-Ig receptor are used to deliver chemotherapeutic agents andcytotoxins to breast cancer and prostate cancer cells, and thereby causethe cancer cells to die. The specific delivery of cytotoxic agents tocancer cells has a long history (Shimizu N (1987) Methods Enzymol 147,382-387). The conceptual basis of this approach is to chemicallyconjugate a cytotoxic protein or compound to an antibody or hormone thatdelivers the toxin-conjugate to cancer cells specifically, therebycausing their death. Very commonly, these agents are linked tomonoclonal antibodies with (relative) specificity for the type of cancertargeted. The monoclonal antibodies usually are directed against cellsurface receptors for hormones or growth factors or other over expressedcell membrane proteins.

Toxin-IgA/IgM/poly Ig Receptor Conjugates are New.

Of the identifiable literature related to toxin conjugates,approximately 50% appears to pertain to cancer related applications ofthis technology, none of which refer to IgA or IgM as vehicles or to thepoly-Ig receptor as a target for toxin conjugates. Although severalextensive reviews of the topic have been published, and many reportshave been published on the status and problems associated with the useof monoclonal antibodies for diagnosis and treatment of cancer, none ofthose references describe the use of IgA, IgM or poly-Ig receptor/toxinconjugates in breast or prostate cancer. Certain toxin conjugates havebeen previously described for breast cancer treatment, including severalbifunctional reagents, and fusion proteins between ligands andantibodies and toxins. A range of protein and compound toxins areavailable, and it is envisioned that one or more of those will besuitable for conjugating to IgA, IgM or the poly-Ig receptor. Apreferred toxic substance for conjugating to IgA, IgM or the poly-Igreceptor topic is an iron-containing compound, suitable for effectingthe delivery of Fe (III) to cells, according to the present method. Asdemonstrated in previous Examples, Fe (III) is a potent cytotoxin forER⁺ breast cancer cells and AR⁺ prostate cancer cells.

ADVANTAGES

Although many different monoclonal antibodies have been developed andused to target both chemical and protein toxins to cancer cells, thepresent approach employs IgA/IgM as the preferred vehicle(s) fordelivery of the toxins and is directed toward a specific target (e.g.,the poly-Ig receptor). The use of the poly-Ig receptor is advantageousbecause that receptor is more localized to secretory/mucosal epithelialtissues that are the primary origins of the major cancers of the bodythan are the other targets that are typically used for targeting oftoxins to cancer cells. The discovery that polymeric IgA and IgMregulate estrogen responsive (ER⁺) breast cancer cells and androgenresponsive (AR⁺) prostate cancer cells has opened new possibilities withregard to targeting the receptor that mediates their function. Sincenon-mucosal cells do not express the poly-Ig receptor, therapeuticmethods that target poly-Ig receptor bearing cancer cells via thesecretory immune system also have certain advantages. One benefit ofsuch a method is that many important organs (e.g., heart and brain) willnot be affected by the treatment.

While the preferred embodiments of the invention have been shown anddescribed, modifications thereof can be made by one skilled in the artwithout departing from the spirit and teachings of the invention. Forexample, one of skill in the art can readily appreciate that for manyapplications, such as estimating risk of cancer, establishing aprognosis, diagnosing, treating, or preventing cancer, a number of themethods, strategies, techniques and compositions described in theExamples may in some cases be advantageously combined, or used inconjunction, to provide a desirable test, treatment or composition.Likewise, in some embodiments, it may be desirable to combine one of thenew methods or compositions with a conventional anti-cancer therapy ortest procedure. The embodiments described herein are exemplary only, andare not intended to be limiting. Many variations and modifications ofthe invention disclosed herein are possible and are within the scope ofthe invention. Accordingly, the scope of protection is not limited bythe description set out above, but is only limited by the claims whichfollow, that scope including all equivalents of the subject matter ofthe claims. Each and every claim is incorporated into the specificationas an embodiment of the present invention. Thus the claims are a furtherdescription and are an, addition to the preferred embodiments of thepresent invention. The disclosures of U.S. Provisional PatentApplication Nos. 60/203,314 filed May 10, 2000; 60/208,348 filed May 31,2000; 60/208,111 filed May 31, 2000; 60/229,071 filed Aug. 30, 2000 and60/231,273 filed Sep. 8, 2000, are hereby incorporated herein byreference. All patents, patent applications and publications citedherein are hereby incorporated herein by reference.

What is claimed is:
 1. A method to aid in diagnosing cancer in amammalian subject comprising: obtaining a population of cells from amucosal epithelial tissue specimen taken from said subject, and (i)determining at least one of a first set of conditions selected from thefollowing: (a) absence of a poly-Ig receptor gene from said cells; or(b) absence of heterozygosity for said poly-Ig receptor gene in saidcells; (ii) determining at least one of a second set of conditionsselected from the following: (c) absence of heterozygosity for a Fcγreceptor gene in said cells; (d) absence of a Fcγ receptor gene fromsaid cells; (e) absence or diminution of a Fcγ receptor in said cells;(f) absence of heterozygosity for a TGF-β receptor gene in said cells;(g) absence of a TGF-β receptor gene from said cells; (h) absence ordiminution of a TGF-β receptor in said cells; or (i) absence or presenceof an estrogen receptor in said cells wherein said absence of at leastone of said first set of conditions or said absence of at least one ofsaid second set of conditions is measured by comparison to similardeterminations in non-neoplastic cells from said subject and/or thesubject's previous test results, or by comparison to a predeterminedstandard value, and wherein an absence of one or more of said first setof conditions and an absence of one or more of said second set ofconditions provides a diagnosis of cancer in said subject.
 2. The methodof claim 1, wherein said first condition is determining the absence of apoly-Ig receptor gene from said cells.
 3. The method of claim 1, whereinsaid first condition is determining the absence of heterozygosity forsaid poly-Ig receptor gene in said cells.
 4. The method of claim 1,wherein the cancer or precancerous lesion is associated with breastcancer, prostate cancer, or colon cancer.
 5. The method of claim 4,wherein said cancer or precancerous lesion is associated with breastcancer.
 6. The method of claim 1, wherein determining the second set ofconditions includes determining the presence of an estrogen receptor insaid cells; wherein said presence is measured by comparison to similardeterminations in non-neoplastic cells from said subject and/or thesubject's previous test results, or by comparison to a predeterminedstandard value, and wherein the presence of at least one of said secondset of conditions provides a diagnosis of cancer in said subject.
 7. Themethod of claim 1, wherein determining the second set of conditionsincludes determining the absence of an estrogen receptor in said cells;wherein said absence is measured by comparison to similar determinationsin non-neoplastic cells from said subject and/or the subject's previoustest results, or by comparison to a predetermined standard value, andwherein the absence of at least one of said second set of conditionsprovides a diagnosis of cancer in said subject.
 8. The method of claim1, wherein one additional condition of the second set of conditions isdetermined.
 9. The method of claim 8, wherein said one additionalcondition is selected from the following: (a) absence of heterozygosityfor a Fcγ receptor gene in said cells; (b) absence of a Fcγ receptorgene from said cells; or (c) absence or diminution of a Fcγ receptor insaid cells.
 10. The method of claim 8, wherein said one additionalcondition is selected from the following: (d) absence of heterozygosityfor a TGF-β receptor gene in said cells; (e) absence of a TGF-β receptorgene from said cells; or (f) absence or diminution of a TGF-β receptorin said cells.
 11. The method of claim 9, wherein said one additionalcondition is: presence of an estrogen receptor in said cells.
 12. Themethod of claim 1, wherein at least two additional conditions aredetermined from the second set of conditions.
 13. The method of claim12, wherein said at least two additional conditions are selected fromthe following: at least one of: (a) absence of heterozygosity for a Fcγreceptor gene in said cells; (b) absence of a Fcγ receptor gene fromsaid cells; or (c) absence or diminution of a Fcγ receptor in saidcells; and at least one of: (d) absence of heterozygosity for a TGF-βreceptor gene in said cells; (e) absence of a TGF-β receptor gene fromsaid cells; (f) absence or diminution of a TGF-β receptor in said cells.14. The method of claim 12, wherein the first of said at least twoadditional conditions are selected from the following: at least one of:(a) absence of heterozygosity for a Fcγ receptor gene in said cells; (b)absence of a Fcγ receptor gene from said cells; or (c) absence ordiminution of a Fcγ receptor in said cells; and the second of at leasttwo additional conditions is: presence of an estrogen receptor in saidcells.
 15. The method of claim 12, wherein the first of said at leasttwo additional conditions are selected from the following: at least oneof: (d) absence of heterozygosity for a TGF-β receptor gene in saidcells; (e) absence of a TGF-β receptor gene from said cells; or (f)absence or diminution of a TGF-β receptor in said cells; and the secondof at least two additional conditions is: presence of an estrogenreceptor in said cells.
 16. The method of claim 12, further comprisingdetermining at least one additional condition selected from thefollowing: (a) absence of heterozygosity for a progesterone receptor insaid cells; (b) absence of a progesterone receptor gene from said cells;(c) absence or diminution of a progesterone receptor in said cells; (d)absence of heterozygosity for insulin-like growth factor-I, or areceptor therefor, in said cells; (e) absence of an insulin-like growthfactor-I gene, or a receptor therefor, from said cells; (f) absence ordiminution of an insulin-like growth factor I, or a receptor therefor,in said cells; (g) absence of heterozygosity for an epidermal growthfactor, or a receptor therefor, in said cells; (h) absence of anepidermal growth factor gene, or a receptor therefor, from said cells;(i) absence or diminution of an epidermal growth factor, or a receptortherefor, in said cells; (j) absence of heterozygosity for a fibroblastgrowth factor, or a receptor therefor, in said cells; (k) absence of afibroblast growth factor gene, or a receptor therefor, from said cells;(l) absence or diminution of a fibroblast growth factor, or a receptortherefor, in said cells; wherein said absence is measured by comparisonto similar determinations in non-neoplastic cells from said subjectand/or the subject's previous test results, or by comparison to apredetermined standard value, and wherein the absence of one or more ofsaid conditions provides a diagnosis of cancer in said subject.
 17. Themethod of claim 12, wherein three additional conditions are determinedfrom said second set of conditions.
 18. The method of claim 17, whereinsaid three additional conditions are selected from the following: atleast one of: (a) absence of heterozygosity for a Fcγ receptor gene insaid cells; (b) absence of a Fcγ receptor gene from said cells; or (c)absence or diminution of a Fcγ receptor in said cells; and at least oneof: (d) absence of heterozygosity for a TGF-β receptor gene in saidcells; (e) absence of a TGF-β receptor gene from said cells; or (f)absence or diminution of a TGF-β receptor in said cells; and (g)presence of an estrogen receptor in said cells.
 19. The method of claim8, wherein said one additional condition comprises: absence of anestrogen receptor in said cells.
 20. The method of claim 19, whereinsaid at least two additional conditions are selected from the following:at least one of: (a) absence of heterozygosity for a Fcγ receptor genein said cells; (b) absence of a Fcγ receptor gene from said cells; or(c) absence or diminution of a Fcγ receptor in said cells; and (g)absence of an estrogen receptor in said cells.
 21. The method of claim19, wherein said at least two additional conditions are selected fromthe following: at least one of: (d) absence of heterozygosity for aTGF-β receptor gene in said cells; (e) absence of a TGF-β receptor genefrom said cells; or (f) absence or diminution of a TGF-β receptor insaid cells; and (g) absence of an estrogen receptor in said cells. 22.The method of claim 19, wherein said three additional conditions areselected from the following: at least one of: (a) absence ofheterozygosity for a Fcγ receptor gene in said cells; (b) absence of aFcγ receptor gene from said cells; or (c) absence or diminution of a Fcγreceptor in said cells; and at least one of: (d) absence ofheterozygosity for a TGF-β receptor gene in said cells; (e) absence of aTGF-β receptor gene from said cells; or (f) absence or diminution of aTGF-β receptor in said cells; and (g) absence of an estrogen receptor insaid cells.